Dendritic cell co-stimulatory molecules

ABSTRACT

A novel costimulatory protein molecule, B7-DC, which is a member of the B7 family, is described as is DNA coding therefor and expression vectors comprising this DNA. B7-DC protein, fragments, fusion polypeptides/proteins and other functional derivatives, and transformed cells expressing B7-DC are useful in vaccine compositions and methods. Compositions and methods are disclosed for inducing potent T cell mediated responses that can be harnessed for anti-tumor and anti-viral immunity.

This application is a continuation of pending U.S. application Ser. No.13/243,966, filed Sep. 23, 2011, entitled “New Dendritic CellCo-Stimulatory Molecules”, by Drew M. Pardoll, Haruo Tsuchiya, Kevin S.Gorski, and Su-Yi Tseng, which is a continuation of U.S. Ser. No.11/931,653, filed Oct. 31, 2007, now U.S. Pat. No. 8,053,558, issuedNov. 8, 2011, which is a continuation of U.S. Ser. No. 11/361,057, filedFeb. 24, 2006, now U.S. Pat. No. 7,560,540, issued Jul. 14, 2009, whichis a continuation of U.S. Ser. No. 09/794,210, filed Feb. 28, 2001, nowU.S. Pat. No. 7,030,219, issued April 18, 2006, which claims benefit ofand priority to U.S. Ser. No. 60/240,169, filed Oct. 13, 2000 and U.S.Ser. No. 60/200,580, filed Apr. 28, 2000, all of which are hereinincorporated in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was funded in part by grants from National Institutes ofHealth, which provides to the United States government certain rights inthis invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention in the field of biochemistry and medicine relates to novelproteins that are selectively expressed on the surface of dendriticcells and can be used as cell surface molecules or in soluble form invaccine compositions to stimulate immune responses.

2. Description of the Background Art

The generation of a T lymphocyte response is a complex process involvingcell-cell interactions and production of soluble mediators (cytokines orlymphokines). This response is regulated by several T-cell surfacemolecules acting as “receptors,” including the T-cell receptor (TCR)complex and other “accessory” surface molecules many of which are cellsurface “differentiation antigens” that were first defined by monoclonalantibodies (“CD molecules”).

Optimal activation of all lymphocytes is believed to require twosignals: an antigen specific or clonal signal, as well as a second,antigen non-specific signal (Janeway, C., Cold Spring Harbor Symp.Quant. Biol. 54:1-14 (1989)). If a lymphocyte encounters an antigenalone, without co-stimulation by so-called co-stimulatory molecules(such as B7 described below), it will respond with either clonalinactivation also called “anergy” (Schwartz, R. Science 248:1349 (1990))or apoptosis (programmed cell death); if the co-stimulatory signal isprovided it will respond with clonal expansion specific for thestimulating antigen. No significant amplification of an immune responseagainst a given antigen occurs without co-stimulation (June et al.(Immunology Today 15:321-331, 1994); Chen et al. (Immunology Today14:483-486); Townsend, S E and Allison, J P (1993) Science 259:368-370).

The quality and potency of an immune response depends in large part onthe type of antigen presenting cells (APC) that process and present theantigen to T cells. The density of the peptide antigen/MHC ligand forengagement of the TCR and the provision of soluble and/or membrane-boundco-stimulatory signals by APCs at the time of T cell engagement andactivation is critical. It is for these reasons that immunotherapeuticstrategies have begun to focus on providing (a) the target antigen tothe appropriate APC types and (b) appropriate co-stimulatory moleculesto enhance T cell activation.

APCs that provide the signals required for activation of T cells includemonocytes/macrophages, B lymphocytes, and, most importantly dendriticcells (DCs). In the past, activated macrophages were believed to be thecritical APCs that initiated T cell responses in vivo. This notion wasbased on their ability to phagocytose antigens effectively and processthem for surface display and presentation. More recently, attention hasshifted to DC as the major initiator in vivo of antigen-specific T cellresponses. DCs have a distinct phenotype from activated macrophages andare classified into different subtypes capable of initiating distinctimmune responses. A functional hallmark of DCs is their approximately100-fold greater potency then macrophages to activate naïve T cells invitro. To date, the explanation of this potency has been based onquantitative differences in molecules known to be important for antigenpresentation. The present invention is based on the discovery of animportant qualitative difference.

The first signal in antigen presentation is initiated by interaction ofthe TCR with antigen presented in the context of class II majorhistocompatibility complex (MHC) molecules on the APC (Allen, Immunol.Today 8:270 (1987)). Co-stimulatory signals come from other molecules,the best characterized of which is the B7 family (namely B7.1, B7.2, andpossibly B7.3) which are also present on APCs

Two proteins expressed on the surface of T cells are thebest-characterized ligands or counter-receptors for co-stimulatorymolecules such as B7. CD28 is a homodimeric glycoprotein of theimmunoglobulin (Ig) superfamily (Aruffo and Seed, Proc. Natl. Acad. Sci.84:8573-8577 (1987)) found on most mature human T cells that functionsin T cell activation. CD28, is constitutively expressed on resting Tcells and increases after activation. After signaling through the T cellreceptor, ligation of CD28 induces T cells to proliferate and secreteIL-2 (Linsley, P S, et al. (1991) J. Exp. Med. 173, 721-730; Gimmi, D D,et al. (1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Thompson, C.B., et al. (1989) Proc. Natl. Acad. Sci. USA. 86, 1333-1337; June, C.H., et al. (1990) Immunol. Today. 11, 211-6; Harding, F. A., et al.(1992) Nature. 357, 607-609.). CD28 mediates cell-cell contact(“intercellular adhesion”), a form of antigen-independent intercellularinteraction that is essential for immune responses (Springer et al. Ann.Rev. Immunol. 5:223-252 (1987)).

CTLA4 is a T cell surface molecule highly homologous to CD28 but is notexpressed on testing T cells and appears following T cell activation(Brunet, J. F., et al., (1987) Nature 328, 267-270). CTLA-4 wasoriginally identified by differential screening of a murine cytolytic Tcell cDNA library, Brunet et al supra. The role of CTLA-4 as a secondreceptor for B7 is discussed in Linsley et al. (1991) J. Exp. Med.174:561-569, which also noted that B7 has a higher affinity for CTLA-4than for CD28. Freeman et al. (1993) Science 262:907-909 discussedCTLA-4 in B7 deficient mice. Ligands for CTLA-4 are described inLenschow et al. (1993) Proc. Nat'l. Acad. Sci. 90:11054-11058.

Th cells secrete growth and differentiation-inducing cytokines such asIL-2, IL-4, and IL-6 possibly in a focused manner in the area of Th-Bcell contact which serves to ensure activation of only B cellspresenting antigen to Th cells and avoid activation of bystander Bcells.

CD28 and CTLA-4 interact with a co-stimulatory molecule generally knownas B7. B7 was originally described as a B cell activation antigenbecause it was found on B cells and was termed B7/BB-1 (Linsley et al.Proc. Natl. Acad. Sci. USA 87:5031-5035 (1990). Hereafter, this moleculewill be referred to as B7, B7-1 or B7.1), B7 and more recently describedB7 homologues are also members of the Ig superfamily. In contrast toCD28 and CTLA-4, B7 comprises two extracellular Ig domains, anN-terminal variable (V)-like domain followed by a constant (C)-likedomain.

B7 family members are generally expressed on APCs and, as noted, are ofcritical importance to the activation of naive T cells. These familymembers include B7-1 (=B7, also designated CD80) and B7-2 (alsodesignated CD86). References describing B7-1 include Schwartz, R. H.Cell 71:1065-1068, 1992; Chen, L., et al. Cell 71:1093-1102, 1992;Freeman, G. J. et al. J. Immunol 143:2714-2722, 1989; and Freeman, G. J.et al. J. Exp. Med. 174:625-631, 1991)). References describing B7-2include (Freeman, G. J. et al. Science 262:909-911 813-960, 1993). Todate, both murine B7-1 and B7-2 and humans B7-1 and B7-2 have beendescribed (Freeman et al., 1989, supra; 1991, supra; and 1993, supra).Activated human B lymphocytes express CTLA4/CD28 bindingcounter-receptors B7-2 and B7-3, both of which can deliver costimulatorysignals to T cells via either CD28 or CTLA4.

B7-2 is expressed by B cells at about 24 hours following stimulationwith either anti-Ig or anti-MHC class II mAbs. B7-2 induces detectableIL-2 secretion and T cell proliferation. At about 48 to 72 hours postactivation, B cells express both B7-1 and a third CTLA4 counter-receptoridentified by a mAB BB-1 (Yokochi T, et al (1982) J. Immunol. 128,823-827), termed B7-3, B7-3 is also expressed on B7-negative activated Bcells and can costimulate T cell proliferation without detectable IL-2production, indicating that the B7-1 and B7-3 molecules are distinct.B7-3 is expressed on a wide variety of cells including activated Bcells, activated monocytes, dendritic cells, Langerhans cells andkeratinocytes. At 72 hours post B cell activation, the expression ofB7-1 and B7-3 begins to decline. The presence of these CTLA4/CD28binding counter-receptors on the surface of activated B lymphocytesindicates that T cell costimulation is regulated, in part, by thetemporal expression of these molecules following B cell activation.

The importance of the B7;CD28/CTLA4 costimulatory pathway(s) has beendemonstrated in vitro and in vivo. A direct relationship exists betweenincreased T cell activity and increased B7 expression (Razi-Wolf et al.,Proc. Natl. Acad. Sci. USA, 89:4210-4214 (1992)). T cells are renderedanergic when they, encounter peptides antigens on cells lacking acostimulatory ligand that binds CD28 Blockade of this costimulatorypathway results in the development of antigen specific tolerance inmurine and humans systems (Harding et al., supra; Lenschow, D. J. et al.(1992) Science, 257, 789-792; Turka, L A et al. (1992) Proc. Natl. Acad.Sci. USA. 89,11102-11105; Gimmi, C D et al. (1993) Proc. Natl. Acad. SciUSA 90, 6586-6590; Boussiotis, V. et al. (1993) J. Exp. Med. 178,1753-1763). Conversely, expression of B7 by B7-negative murine tumorcells induces T-cell mediated specific immunity accompanied by tumorrejection and long lasting protection to tumor challenge (Chen, L., etal. (1992) Cell 71:1093-1102; Townsend et al., supra; Baskar, S., et al.(1993) Proc. Natl. Acad. Sci. 90, 5687-5690). Therefore, manipulation ofthe B7:CD28/CTLA4 pathway offers great potential to stimulate orsuppress immune responses in humans.

Interactions between CB28 and B7 have been characterized using geneticfusions of the extracellular portions of B7 or CD28 with Ig Cγ1 chains(Linsley et al, J. Exp. Med. 173:721-730 (1991). When B7Ig fusionproteins are immobilized, or when B7 is expressed on the surface of acell, such as a transfected CHO cell, they costimulate T cellproliferation. T cell stimulation with B7+ CHO cells also specificallystimulates increased levels of transcripts for IL-2.

U.S. Pat. No. 5,522,288 describes a method for regulating immuneresponses by contacting CD28 positive T cells with fragments encoded byparts of DNA encoding B7, primarily corresponding to the extracellulardomain (BCD) of B7. Immune responses were also legislated by derivativesof B7 that were are fusion protein constructs including at least aportion of B7 ECD and another protein, such as the human IgCγ1 domainthat altered the solubility, binding affinity and/or valency of B7. Forexample DNA encoding amino acid residues from positions 1-215 of the B7ECD was joined to DNA encoding amino acid residues of the sequencescorresponding to the hinge, CH2 and CH3 regions of human IgCγ1 to form aDNA fusion product which encoded a B7Ig fusion protein. Also disclosedwas a method for treating an immune system disease mediated by T cellsby administering B7 or B7Ig fusion protein to react with T cells bybinding the CD28 receptor. T cell proliferation in graft versus hostdisease was inhibited by reacting CD28+ T cells with B7 antigen or B7Igfusion protein in combination with an immunosuppressant.

U.S. Pat. No. 5,861,310 discloses tumor cells modified to express one ormore T cell costimulatory molecules, including B7-2 and B7-3. Oneembodiment includes further expression of B7. Modification was bytransfection with nucleic acid encoding the B7-2, B7-3 or B7 proteins.Tumor cells could also be genetically modified in vivo. Such modifiedtumor cells said to be useful for treating a patient with a tumor, toprevent or inhibit metastatic spread or inhibit recurrence of the tumor.This document disclosed a method for specifically inducing a CD4+ T cellresponse against a tumor.

U.S. Pat. No. 5,942,607 discloses isolated nucleic acids encoding novelCTLA4/CD28 ligands which costimulate T cell activation. In oneembodiment, the isolated nucleic acid encoded B7-2. Also disclosed was anucleic acid comprising at least a portion of the disclosed full lengthB7-2 sequence. According to this document, the nucleic acid sequencescould be integrated into various expression vectors which could directthe synthesis of the corresponding proteins or peptides in a variety ofhost cells including mammalian and insect cells. Also disclosed warehost cells transformed to produce proteins or peptides encoded by thesenucleic acid sequences and isolated proteins and peptides which compriseat least a portion of the B7-2 sequence.

Dong H et al., Nat Med 1999 5:1365-1399, described a third member of theB7 family, designated B7-H1 that does not bind CD28, CTLA4 or ICOS(inducible co-stimulator). Ligation of B7-H1 co-stimulated T-cellresponses to polyclonal stimuli and alloantigens, and preferentiallystimulated the production of interleukin-10, IL-2, produced in smallamounts, was required for the effect of B7-H1 co-stimulation. This studydefined a previously unknown co-stimulatory molecule that may beinvolved in the negative regulation of cell-mediated immune responses.The same laboratory (Wang S et al, Blood. 2000; 96:2808-2813) describeda new human B7-like gene designated B7-H2, the expression of which wasdetected on the surface of monocyte-derived immature DCs. Soluble B7-H2and an Ig fusion protein, B7-H2Ig, bound to activated, but not resting,T cells. This binding was inhibited by a soluble form of ICOS (ICOSIg)but not by CTLA4Ig. ICOSIg stained CHO cells transfected with the B7-H2gene. Using suboptimal cross-linking of CD3 as a stimulus, costimulationof T-cell proliferation by B7-H2Ig was found to be dose-dependent andcorrected with secretion of IL-2, whereas optimal CD3 ligationpreferentially simulated IL-10 production. The authors concluded thatB7-H2 is a putative ligand for the ICOS T-cell molecule.

Swallow M M et al., Immunity, 1999, 11:423-432 reported cloning of anovel gene, b7h, a is a close homolog of B7 molecules that are expressedon APCs. B7h costimulated proliferation of purified T cells by acting ona receptor distinct from CD28 or CTLA-4. Surprisingly, although B7h wasexpressed in unstimulated B cells, its expression was induced innonlymphoid cells (3T3 cells; embryonic fibroblasts) treated with TNFαand was upregulated in nonlymphoid tissue of mice treated with LPS, apotent activator of TNFα. These studies defined a novel costimulatoryligand of cells and suggested that induction of B7h by TNFα may directlyaugment recognition of self during inflammation

Yoshinaga S K et al., Nature, 1999, 402:827-832, described a new murinecostimulatory receptor-ligand pair. The receptor, related to CD28, wasthe murine homologue of the human protein ICOS, and was expressed onactivated T cells and resting memory T cells. The ligand, which washomologous to B7 molecules was designated B7-related protein-1 (B7RP-1).B7RP-1 is a type 1 transmembrane protein with 20% and 19% amino acididentity to murine B7.1 (CD80) and B7.2 (CD86), respectively. Thishomology is significant as B7.1 and B7.2 share only 27% amino acididentity (Freeman, G J et al., J. Exp. Med. 178:2185-2192 (1993)). Thishomology includes the cysteines that are important for Ig loop formationat conserved locations (residues 62, 138,185 and 242 from the initiatingmethionine). The overall length and relative position of thetransmembrane domain of B7RP-1 are similar to those of the B7 molecules(Greenfield, E A et al., Crit. Rev. Immunol. 18:389-418 (1998)). B7RP-1was shown to be expressed on B cells and macrophages. ICOS and B7RP-Idid not interact with proteins in the CD28-B7 pathway, and B7RP-1co-stimulated T cells independently of CD28. Transgenic mice expressinga fusion protein between B7RP-1 and the Fc portion of Ig (“B7-RP1-Fc”)had lymphoid hyperplasia in spleen, lymph nodes and Peyer's patches. Costimulatory activity of B7RP-1 in vivo was found by demonstratingenhanced delayed-type hypersensitivity in antigen-presensitized micetreated with B7RP-1-Fc at the time of antigen challenge. The authorsconcluded that ICOS and B7RP-1 define a distinct new receptor-ligandpair that is structurally related to CD28-B7 and is involved in theadaptive immune response.

Yoshinaga S K et al., Int Immunol, 2000 October 12:1439 -4447 , reportedco-stimulation of human T cells through the human B7RP-1 and ICOSinteraction. This ligand-receptor pair interacted with a K_(D) ofapproximately 33 nM and an off-rate having a t_((1/2)) of >10 min. TNFαdifferentially regulated expression of human B7RP-1 on B cells,monocytes and DC. TNFα enhanced B7RP-1 expression on B cells andmonocytes, but inhibits expression on DC. A human B7RP-1 -Fc protein, orcells that expressed membrane-bound B7RP-1, co-stimulated T cellproliferation in vitro. Specific cytokines, such as IFNγ and IL-10, wereinduced by B7RP-1 co-stimulation. Although IL-2 levels were notsignificantly increased, B7RP-1 -induced co-stimulation was dependent onIL-2. These studies defined the human ortholog to murine B7RP-1 andcharacterized its interaction with human ICOS.

PD-1 is an immuno-inhibitor receptor expressed by activated T, B andmyeloid cells. Mice deficient in PD-1 showed multiple forms ofautoimmunity due to the loss of peripheral tolerance, Freeman, G J etal., J. Exp. Med. 192:1027-1034 (2000) reported that the ligand of PD-1(PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1resulted in inhibition of TCR-mediated lymphocyte activation(proliferation, cytokine secretion). In addition, PD-1 signalinginhibited suboptimal levels of CD28-mediated costimulation. PD-L1 isexpressed by APCs (human monocytes stimulated with IFNγ, activated humanDCs). In addition, PD-L1 was shown to be expressed in heart and lung.The authors speculated that relative magnitude of inhibitor PD-L1signals and costimulatory B7-1/B7-2 signals on APCs may determine theextent of T cell activation and the threshold between tolerance andautoimmunity. The presence of PD-L1 on nonlymphoid tissues maycontribute to the magnitude of immune responses at sites ofinflammation.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

SUMMARY OF THE INVENTION

In order to identify genes encoding novel dendritic cell (DC) specificcostimulatory molecules for T cell activation, the investors screened asubtracted c-DNA library between DCs and activated macrophages. ThiscDNA subtraction approach defines genes expressed by DCs but not byactivated macrophages. Use of this approach has led to discover ofseveral novel DC-specific genes that are useful in enhancing the potencyof vaccines that depend on activation of T cells. The presentapplication focuses on one such gene.

Based on presence in the DC library and absence from the activatedmacrophage library, a novel coding sequence, termed “B7-DC” wasidentified. The B7-DC gene is a member of the B7 family of genesencoding costimulatory molecules. B7-DC is the first B7 family memberwith DC-specific expression and different receptor specificity. Theproduct of this gene has an important role in mediating the uniqueability of DCs to stimulate T cells. Functional analysis indicated thatB7-DC is more active than B7-1 in stimulating IFNγ production by Tcells. B7-DC DNA and polypeptides are therefore useful in compositionsand methods to enhance the efficacy of cellular and molecular vaccinecompositions, whether antigen-specific or not.

In one embodiment, the present invention provides an isolated nucleicacid molecule that encodes a mammalian protein termed B7-DC that isselectively expressed on dendritic cells as compared to activatedmacrophages. The nucleic acid molecule preferably comprises a nucleotidesequence selected from SEQ ID NO:1 (of human origin) or SEQ ID NO:5. (ofmurine origin). The invention is also directed to an isolated nucleicacid that hybridizes with the above nucleic acid molecule understringent hybridization conditions. Preferred stringent conditionsinclude incubation in 6×sodium citrate (SSC) at about 45° C., followedby a wash in about 0.2×SSC at a temperature of about 50° C. Preferablythe above nucleic acid molecule comprises the nucleotide sequence SEQ IDNO:1. A preferred nucleic acid molecule as above encodes a proteinhaving an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:4or encodes a biologically active fragment, homologue or other functionalderivative of the protein. Preferably, the nucleic acid molecule encodesthe protein having the sequence SEQ ID NO:2 (B7-DC of human origin) orencodes the biologically active fragment, homologue or other functionalderivative of SEQ ID NO:2.

In a preferred embodiment, the nucleic acid molecule encodes theextracellular domain of the B7-DC protein, which includes residues26-221, which encodes a co-stimulatory homologue, fragment or otherfunctional derivative thereof.

In another embodiment, the above nucleic acid molecule of encodes B7-DCfusion protein which comprises:

-   (a) a first nucleic acid sequence encoding a first polypeptide that    is all or a part of a B7-DC protein (preferably SEQ ID NO:2 or SEQ    ID NO:4);-   (b) optionally, fused in frame with the first nucleic acid sequence    a linker nucleic acid sequence encoding a linker peptide; and-   (c) a second nucleic acid sequence that is linked in frame to the    first nucleic acid sequence or to the linker nucleic acid sequence    and that encodes a second polypeptide.    The second polypeptide preferably consists of one or more domains of    an Ig heavy chain constant region, preferably the two C domains of    human IgG, preferably IgG1.

Also provided is an expression vector comprising any of the abovenucleic acid molecules operatively linked to

-   (a) a promoter and-   (b) optically, additional regulatory sequences that regulate    expression of the nucleic acid in a eukaryotic cell.

The above expression vector may be a plasmid or a viral vector. Thesevectors include self replicating RNA replicons (DNA-launched or RNA),suicide RNA vectors DNA viruses (such as adenovirus, vaccina virus,etc.) and RNA virions grown on packaging cell lines.

The vector DNA or RNA may be complexed to gold particles for genegun-mediated introduction to a host or complexed with other polymers,for example, in controlled release formulations, that enhance deliveryto the desired target cells and tissues.

Also included is a vector composition which comprises:

-   (a) a first recombinant expression vector having incorporated in its    sequence a nucleotide sequence encoding an antigen of interest    against which an immune response is to be induced; and-   (b) a second recombinant expression vector having incorporated in    its nucleic acid sequence or more nucleotide sequences encoding a    co-stimulator polypeptide, at least one of which polypeptides is    B7-DC, or a biologically active fragment, homologue or other    functional derivative thereof,    wherein the expression vectors are able to co-infect or co-transfect    a host cell resulting in co-expression of the antigen and the    costimulator polypeptide, fragment, homologue or derivative.

In a modification of the above embodiment, the invention provides athird nucleic sequence encoding a targeting protein that (i) promotesspread of the expressed product (antigen) between cells, preferablyAPCs, (ii) increases the display of the antigen on APCs in which thenucleic acid is expressed, and/or (iii) promotes the representation(cross-priming) and display of the antigen in APCs of a host into whichthe vector is introduced. The targeting protein-encoding nucleic acidmay be fused to the nucleic acid encoding the antigen or theco-stimulator or both. acid the first or the second vector includesnucleic acid. In one embodiment, the vector composition combines theeantigen-encoding nucleic acid, the co-stimulator-encoding nucleic acid(preferably B7-DC) and a “targeting” protein-encoding nucleic acid intoa single fused construct.

This invention includes a cell transformed or transfected with any ofthe above nucleic acid molecules or expression vectors. The cell ispreferably a eukaryotic cell, more preferably a mammalian cell, mostpreferably a human cell. The cell may be a dendritic cell or aprogenitor thereof. In another embodiment, the cell is a tumor cell,preferably a tumor cell that bears an antigen that is the same as, orcross-reactive with, an antigen on a tumor in the host against which animmune response is desired.

A preferred embodiment is an isolated mammalian tumor cell transfectedwith an exogenous nucleic acid molecule encoding a mammalian B7-DCprotein (preferably SEQ ID NO:2 or SEQ ID NO:4) or a biologically activefragment, homologue or other functional derivative thereof, such thatwhen the protein, fragment, homologue or derivative is expressed by thetumor cell and the tumor cell is contacted with T cells

-   (i) the B7-DC protein, fragment, homologue or derivative binds to    the T cells; and-   (ii) the tumor cell costimulates the T cells to proliferate and/or    to produce and secrete cytokines.

The present invention is also directed to a polypeptide that isselectively expressed on dendritic cells as compared to activatedmacrophages and has the following functional properties:

-   (a) binds to a binding partner on T cells; and-   (b) costimulates T cells to proliferate and/or to produce and    secrete cytokines.

Also included are biologically active fragments, homologues or otherfunctional derivatives of the polypeptide.

The polypeptide, fragment, homologues or fractional derivative ispreferably encoded by a nucleic acid molecule having the sequence SEQ IDNO:1 or SEQ ID NO:5, or a fragment homologue or equivalent of thenucleic acid molecule. A preferred polypeptide has the ammo acidsequence SEQ ID NO:2 or SEQ ID NO:4.

The polypeptide or a biologically active fragment, homologues or otherfunctional derivative of the polypeptide may be produced by recombinantexpression of one of the above nucleic acids.

A preferred polypeptide comprises the extracellular domain of the B7-DCprotein, preferably

-   (a) amino acid residues 26-221 of SEQ ID NO:2 (human) or-   (b) amino acid residues 26-221 of SEQ ID NO:4 (mouse).    The above polypeptide may consist essentially of the extracellular    domain of B7-DC

Also provided is a B7-DC fusion polypeptide having a first fusionpartner comprising all or a part of a B7-DC protein fused

-   (i) directly to a second polypeptide or,-   (ii) optionally, fused to a linker peptide sequence that is fused to    the second polypeptide.

The above A B7-DC fusion protein may also be fused to a secondpolypeptide, preferably one of more domains of an Ig heavy chainconstant region, preferably having an amino acid sequence correspondingto the hinge, C_(H)2 and C_(H)3 regions of a human immunoglobulin Cγ1chain.

In one embodiment of the above fusion protein, the first fusion partneris the extracellular domain of a B7-DC protein the full length sequenceof which is SEQ ID NO:2 or SEQ ID NO:4.

The fusion protein preferably hinds to a binding partner on T cells andco-stimulates T cells in the presence of an adequate stimulus to the Tcell receptor.

Also provided is a dimeric or trimeric fusion protein which is a dimeror trimer of the above fusion proteins. Preferably, the chains aretandemly linked via disulfide bonds or other interchain covalent bonds.

In a preferred dimeric fusion protein, the dimer results from thecovalent bonding of Cys residue in the C_(H) regions of two of the Igheavy chains that are the same Cys residues that are disulfide linked indimerized normal Ig H chains.

The fusion protein of the invention may comprise a multimer of two ormore repeats of the first fusion partner linked end to end, directly orwith a linker sequence between one or more monomers.

The present invention also provides an antibody that is specific for anepitope of a B7-DC protein, which epitope is not present in a knownmember of a B7 family protein. The epitope may be a linear orconformational epitope of a polypeptide of SEQ ID NO:2 or SEQ ID NO:4.The antibody is preferably a monoclonal antibody, more preferably ahuman or humanized (via engineering) monoclonal antibody.

Also provided is a method of using the above antibody to identify orquantitate cells expressing a B7-DC polypeptide on their surface in acell population, comprising

-   (a) contacting cells of the population with the above antibody so    that the antibody binds to cells expressing the epitope;-   (b) assessing the presence of or quantitating the number of cells to    which the antibody is bound.

Another method is provided for isolating cells expressing a B7-DCpolypeptide on their surface from a cell population, comprising

-   (a) contacting the population with the above antibody so that the    antibody binds to cells expressing the epitope;-   (b) positively selecting cells to which the antibody has bound or    negatively selecting cells to which the antibody has not bound.

Also provided is a method of detecting the presence or quantitating aB7-DC polypeptide, fragment or homologue in a sample, comprising thesteps of:

-   (a) contacting the sample with the antibody of claim 43 such that    the antibody binds to any polypeptides or fragments bearing the    epitope;-   (b) detecting the presence of, or quantitating the polypeptides or    fragments bound to the antibody.

The present invention is also directed in a method of inducing orincreasing the expression of a B7-DC polypeptide in an antigenpresenting cell or a progenitor thereof to increase the ability of thecell to co-stimulate T cells in vitro or in vivo in the presence of anadequate stimulus to the T cell receptor, comprising transforming ortransfecting the antigen presenting cell or progenitor cell with theexpression vector in described above, such that the expression of theB7-DC polypeptide is induced or increased on the cells. The antigenpresenting cells are preferably dendritic cells and the progenitors aredendritic poll progenitors.

The present invention provides method for simulating immune responsesusing cellular co-stimulatory compositions as well as polypeptideco-stimulators. One method for increasing the T cell response of amammalian subject to antigenic stimulation comprises administering tothe subject an effective amount of cells as described above, preferablytumor cells, in conjunction with an antigenic stimulus, wherein thecells are effective to increase the T cell response of the subject tothe antigenic simulation. The foregoing is preferably accomplished byco-injection of the antigen and the co-stimulatory composition.

A method for increasing the T cell response of a mammalian subject toantigenic stimulation with a tumor-associated antigen, comprisesadministering to the subject an effective amount of tumor cells asdescribed above, wherein the tumor cells express the antigen, theadministration of the tumor cells being effective to increase the T cellresponse of the subject to the tumor antigen stimulation.

A method for increasing the T cell response of a mammalian subject toantigenic stimulation, comprising administering to the subject ineffective amount of a polypeptide, fragment, homologue or functionalderivative as above, or a fusion polypeptide or protein as above, inconjunction with an antigenic stimulus, wherein the administration ofthe polypeptide is effective to increase the T cell response of thesubject to the antigenic stimulation.

This invention also provides a method for inhibiting a T cell responseof a mammalian subject to antigenic stimulation, comprisingadministering to the subject an effective amount of an antibody asdescribed, wherein the administration of the antibody is effective toblock stimulation of T cells or to eliminate antigen-reactive T cells,thereby inhibiting the T cell response. These methods are particularlyuseful for treating a subject with a tissue or organ transplant toinhibit transplant rejection and/or to promote engraftment. In the caseof an autoantigen, the method blocks or diminishes autoimmune reactionsand their pathologic sequelae.

The present invention provides therapeutic methods using T cells thathave undergone ex vivo stimulation with the compositions of thisinvention. One method for increasing the immune response of a mammaliansubject to antigenic stimulation comprises:

-   (a) obtaining T cells from the subject, from an immunologically    compatible donor for said subject, or from an immunologically    acceptable cultured cell line;-   (b) contacting the T cells ex vivo with an effective amount of cells    as described above, wherein the contacting is effective to increase    the response of the T cells to antigenic stimulation; and-   (c) administering the T cells of step (b) to the subject,    thereby increasing the immune response of the subject.

In another embodiment, the method for increasing the immune response ofa mammalian subject to antigenic stimulation comprises:

-   (a) obtaining T cells from obtaining T cells from the subject, from    an immunologically compatible donor for said subject, or from an    immunologically acceptable cultured cell line;-   (b) contacting the T cells ex vivo with an effective amount of (i) a    polypeptide, fragment, homologue or functional derivative as    described above, or (ii) a fusion polypeptide as above, wherein the    contacting is effective to increase the response of the T cells to    antigenic stimulation; and-   (c) administering the T cells of step (b) to the subject,    thereby increasing (or generating) an immune response of the    subject.

Also provided herein is a vaccine composition comprising

-   (a) (i) cells as described above that express a B7-DC    construct, (ii) a B7-DC polypeptide, fragment, homologue or    functional derivative, (iii) a B7-DC fusion polypeptide or protein-   (b) generally an additional source of antigen to which an immune    response is desired—though this may not be required in the case of    the cell-based vaccine wherein the cells themselves expresses the    antigen (as in the case of tumor antigen-bearing tumor cells);-   (c) optionally, a general immunostimulatory agent or adjuvant; and-   (d) a pharmaceutically and immunologically acceptable excipient or    carrier for (a), b) and (c).

A method for inducing or enhancing an immune response to an antigen to amammalian subject comprise administering to the subject an effectiveamount of the above vaccine composition.

Also provided is a co-stimulatory composition for use with an antigen ora vaccine, comprising:

-   (a) a B7-DC polypeptide (preferably SEQ ID NO: 2 or SEQ ID NQ:4), a    fragment, a homologue or a functional derivative thereof, or a B7-DC    fusion polypeptide, and-   (b) a pharmaceutically and immunologically acceptable excipient or    carrier.

A method for potentiating an immune response to an antigen or a vaccinein a mammalian subject, comprises administering to the subject, incombination with the antigen or vaccine, the above costimulatorycomposition.

A method of stimulating a systemic immune response to a tumor in asubject, comprises administering to the subject genetically alteredtumor cells which cells

-   (a) are derived from a tumor in the subject, and-   (b) are genetically altered by introduction ex vivo of a B7-DC    nucleic acid as described above, the expression of which provides a    costimulatory signal in the subject,    wherein the administering results in stimulation of the systemic    immune response directed to the tumor.

The tumor cells are preferably treated, preferably by irradiation, toprevent their growth after they have bees administered.

The subject may be subjected to a tumor-reducing regimen ofchemotherapy, irradiation or surgical resection prior to theadministering of the above therapeutic compositions.

Also provided is a method of inducing an antitumor response in a mammalhaving an antigen-positive tumor, comprising:

-   (a) providing cells of the tumor or of a tumor cell line that    -   (i) express antigens shared with the tumor of the mammal;    -   (ii) are transfected with a B7-DC-encoding nucleic acid vector        as above, that when expressed, s a B7-DC molecule causes the        cells to co-stimulate a T cell response to antigens of the        tumor;    -   (iii) optionally, are irradiated prior to step (b);-   (b) administering an effective number of the cells to the mammal,    which cells express the antigens and the B7-DC molecule;    thereby inducing an antitumor response

In the above method, the antitumor response is characterized by:

-   -   (A) at least a 50% decrease in the sum of the products of        maximal perpendicular diameters of all measurable lesions;    -   (B) no evidence of new lesions, and    -   (C) no progression of any preexisting lesions.

Also provided is a method of inducing regression or attenuation ofprimary growth or regrowth of a tumor in a mammal bearing the tumor,comprising:

-   (a) providing cells of the tumor or of a tumor cell line that    -   (i) express antigens shared with the tumor of the mammal;    -   (ii) are transferred with a B7-DC-encoding nucleic acid vector        as above, that, when expressed, as a B7-DC molecule causes the        cells to co-stimulate a T cell response to antigens of the        tumor;    -   (iii) optionally, are irradiated prior to step (b);-   (b) administering an effective number of the cells to the mammal,    which cells express the antigens and the B7-DC molecule;    thereby inducing a systemic immune response specific to the tumor    antigens of the melanoma, thereby inducing the regression or the    attenuation.

A method of inhibiting recurrent growth of an antigen-positive tumor ina mammal, comprises:

-   (a) providing cells of the tumor or of a tumor cell line that    -   (i) express antigens shared with the tumor of the mammal;    -   (ii) are transfected with a B7-DC-encoding nucleic acid vector        as above, that, when expressed, causes the cells to co-stimulate        a T cell response to antigens of the tumor;    -   (iii) optionally, are irradiated prior to step (b);-   (b) administering an effective number of the cells to the mammal,    which cells express the antigens and the B7-DC molecule;    thereby inducing a systemic immune specific to the tumor antigens in    the mammal, which immune response inhibits the recurrent growth of    the tumor.

Another embodiment is directed to a method of providing a co-stimulatorysignal in the vicinity of locally-administered antigen in a mammaliansubject to promote the local generation of an inflammatory and immuneresponse that results in a state of systemic immunity to the antigen,the method comprising administering to a local site in the subject

-   (a) cells that express a costimulation-effective amount of B7-DC    polypeptide, fragment, homologue or functional derivative as above,    and    -   (b) the antigen        such that costimulation in physical proximity with the antigen        promotes the local generation of the response and results in the        state of systemic immunity.

In the above method, the antigen is preferably a tumor antigen that isadministered in (b) in the form of tumor cells or subcellular antigenicmaterial. The tumor cells may also be the cells that express the B7-DCpolypeptide, fragment, homologue or derivative in (a).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing shows the map of hB7-DC which is localizedon human chromosome 9p24. hB7-DC maps to BAC clone RPCI-11.2.

FIG. 2 shows that B7-DC is differentially expressed between DCs andmacrophages. Distribution of B7-DC mRNA in bone marrow DCs, splenic DCs,macrophages, macrophage lines and tissues was assessed by virtualNorthern blot analysis using 0.5 μg/lane purified DNA run on a 1%agarose gel. G3PDH was used as control J774A1, Raw264.7, Pu5-1.8 andWEHI cells are macrophage cell lines. BM: bone marrow.

FIG. 3 shows a virtual Norton blot of B7-DC expression on human DCs.Lane 1 shows human DCs cultured with GM-CSF+Flt-3L, lane 2 shows humanplacenta and lane 3 shows human DCs cultured with GM-CSF+IL4.Oligonucleotides from the 5′ and 3′ UTR of human B7-DC were used to makePCR DNA probe for virtual Northern analysis of total RNA of human DCs.β-actin was used as control to ensure the quality of mRNA.

FIG. 4 represents is a flow cytometric analysis showing surfaceexpression of B7-DC on mature BM-DCs. Day 9 murine BM-DCs wereFe-blocked and stained with control antibody or B7-DC antisera.Specificity of binding was demonstrated by adding B7-DC-Ig to competefor the binding of anti-B7-DC to the surface of DCs.

FIG. 5 shows the binding of B7-DC to PD-1 but not CTLA-4 or CD28. 293Tcells were transiently transfecfed with pCAGGS-B7.1 o pCAGGS-B7-DC.Transfectants were stained with PD-1-IG, 28-IG and CTLA-4-Ig fusionmolecules followed by PE-labeled secondary antibody. Staining of pCAGGS(empty vector) transfectants was negative (not shown)

FIG. 6 (let and right panel) shows the costimulation of T cellproliferation by anti-CB3 and B7-DC-Ig. Left graph: purified T cells(CD4+CD8) were cultured in wells pre-coated with increasingconcentrations of anti-CD3 (mAb 2C11) and a fixed concentration (0.1μg/ml) of immobilized B7.1-Ig (♦), B7-DC-Ig (●) or isotype control (▴).Results depict one representative experiment of three. Cells wereincubated for 72 h and labeled with ³H-thymidine. CPM, counts perminute. Right Graph: purified CD8 T cells were cultured in wellspre-coated with increasing concentrations of anti-CD3 and fixedconcentration of immobilized B7.1-Ig (♦), B7-DC-Ig (●) or isotypecontrol (▴) as in (a). Results are of one representative experiment oftwo. Cells were incubated for 72 h and labeled with ³H-thymidine. CPM,counts per minute.

FIG. 7 shows the costimulation of antigen-specific T cell proliferativeresponses RENCA cells were treated with IFNγ for 72 hrs to induce MHCclass II expression and incubated with 12.5 μg/ml of HA110-120 peptide.Purified HA+I-E^(d) specific transgenic T cells were added together withincreasing concentrations of either B7.1-Ig (♦), B7-DC-Ig (●) or Isotypecontrol (▴) in soluble form. Cells were incubated for 48 h and labeledwith ³H-thymidine. CPM, counts per minute. Results are onerepresentative experiment of three.

FIG. 8 shows cytokine secretion of T cells costimulated by B7-DC. Upperpanels: Purified T cells were cultured in wells pre-coated with anti-CD3(0.12 μg/ml) and 0.1 μg/ml of immobilized B7.1-Ig (♦), B7-DC-Ig (●) orisotype control (▴) as in FIG. 6 (left). Results depict onerepresentative experiment of three. Lower panels: γ-IFN treated RENCAcells loaded with 12.5 μg/ml HA(110-120) peptide were incubated withpurified HA+I-E^(d) specific transgenic T cells together with 2 μg/ml ofsoluble B7.1-Ig, B7-DC-Ig or isotype control (symbols as above). Resultsdepict one representative experiment of two. Supernatants were collectedafter 24 h and 48 h culture and assayed for the indicated lymphokinesusing ELISA.

FIG. 9 shows that B7-DC-Ig greatly enhances antigen specificproliferation after in vivo co-stimulation. After adoptive transfer of2.5×10⁶ TCR transgenic cells specific for HA, three groups of mice wereimmunized s.c., in their hind footpads with either HA peptide (110-120),incomplete Freund's adjuvant (IFA) alone or in combination with eitherB7-DC-Ig+IFA or an isotype control antibody with IFA. Draining lymphnodes were harvested on day 7. 1.5×10⁵ LN cells were incubated with theHA peptide for 48 h, pulsed with 1 μCi[³H]thymidine and theradioactivity incorporated after 12 h was determined.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have now identified new proteins and nucleic acidsthat serve as the basis for improved immunotherapeutic compositions andmethods. Human and murine forms of a novel costimulatory protein namedB7-DC have been discovered and are disclosed herein.

DNA encoding human B7-DC has the nucleotide sequence SEQ ID NO:1, showbelow.

1 atgatcttcctcctgctaatgttgagcctggaattgcagcttcaccagatagcagcttta 61ttcacagtgacagtccctaaggaactgtacataatagagcatggcagcaatgtgaccctg 121gaatgcaactttgacactggaagtcatgtgaaccttggagcaataacagccagtttgcaa 181aaggtggaaaatgatacatccccacaccgtgaaagagccactttgctggaggagcagctg 241cccctagggaaggcctcgttccacatacctcaagtccaagtgagggacgaaggacagtac 301caatgcataatcatctatggggtcgcctgggactacaagtacctgactctgaaagtcaaa 361gcttcctacaggaaaataaacactcacatcctaaaggttccagaaacagatgaggtagag 421ctcacctgccaggctacaggttatcctctggcagaagtatcctggccaaacgtcagcgtt 481cctgccaacaccagccactccaggacccctgaaggcctctaccaggtcaccagtgttctg 541cgcctaaagccaccccctggcagaaacttcagctgtgtgttctggaatactcacgtgagg 601gaacttactttggccagcattgaccttcaaagtcagatggaacccaggacccatccaact 661tggctgcttcacattttcatcccctcctgcatcattgctttcattttcatagccacagtg 721atagccctaagaaaacaactctgtcaaaagctgtattcttcaaaagacacaacaaaaaga 781cctgtcaccacaacaaagagggaagtgaacagtgctatc 819

The human B7-DC protein has the amino acid sequence SEQ ID NO: 2, shownbelow (with leader sequence, transmembrane domain and cytoplasmic tailannotated):

    putative leader sequence 1MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTL ECNFDTGSHV 50 51NLGAITASLQ KVENDTSPHR ERATLLESQL PLGKASFHIP QVQVRDEGQY 100 101QCIIIYGVAW DYKYLTLKVK ASYRKINTHI LKVPETDEVE LTCQATGYPL 150 151AEVSWPNVSV PANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVR 200                           putative TM doamin 201ELTLASIDLQ SQMEPRTHPT WLLHIFIPSC IIAFIFIATV  IAL RKQLCQKL 250 251LYSSKDTTKR PVTTTKREVN SAI 273      cytoplasmic tailThe extracellular domain of this protein is from residue P²⁶ throughresidue W²²¹.

A DNA clone that includes the coding sequence encoding murine B7-DC hasthe nucleotide sequence SEQ ID NO:3, show below. The coding sequence(underscored, set off in triplets) begins from the methionine codon(bolded) beginning at nucleotide 210 and terminates with the tag stopcodon (bolded) (nucleotides 951-953)

gaattcggcacgaggtcaaatgtggcatatctttgttgtctccttctgtctcccaactag 60agagaacacacttacggctcctgtcccgggcaggtttggttgtcggtgtgattggcttcc 120agggaacctgatacaaggagcaactgtgtgctgccttttctgtgtctttgcttgaggagc 180tgtgctgggtgctgatattgacacagacc 209atg ctg ctc ctg ctg ccg ata ctg aac ctg agc tta caa ctt cat cct 257gta gca gct tta ttc acc gtg aca gcc cct aaa gaa gtg tac acc gta 305gac gtc ggc agc agt gtg agc ctg gag tgc gat ttt gac cgc aga gaa 353tgc act gaa ctg gaa ggg ata aga gcc agt ttg cag aag gta gaa aat 401gat acg tct ctg caa agt gaa aga gcc acc ctg ctg gag gag cag ctg 449ccc ctg gga aag gct ttg ttc cac atc cct agt gtc caa gtg aga gat 497tcc ggg cag tac cgt tgc ctg gtc atc tgc ggg gcc gcc tgg gac tac 545aag tac ctg acg gtg aaa gtc aaa gct tct tac atg agg ata gac act 593agg atc ctg gag gtt cca ggt aca ggg gag gtg cag ctt acc tgc cag 641gct aga ggt tat ccc cta gca gaa gtg tcc tgg caa aat gtc agt gtt 689cct gcc aac acc agc cac atc agg acc ccc gaa ggc ctc tac cag gtc 737acc agt gtt ctg cgc ctc aag cct cag cct agc aga aac ttc agc tgc 785atg ttc tgg aat gct cac atg aag gag ctg act tca gcc atc att gac 833cct ctg agt cgg atg gaa ccc aaa gtc ccc aga acg tgg cca ctt cat 881gtt ttc atc ccg gcc tgc acc atc gct ttg atc ttc ctg gcc ata gtg 929ata atc cag aga aag agg atc tag 953gggaagctgtattacggaagaagtggtctcttcttcccagatctggacctgcggtcttgg 1013gagttggaaggatctgatgggaaaccctcaagagacttctggactcaaagtgagaatctt 1073gcaggacctgccatttgcacttttgaaccctttggacggtgacccagggctccgaagagg 1133agcttgtaagactgacaatcttccctctgtctcaagactctctgaacagcaagaccccaa 1193tggcactttagacttacccctgggatcctggaccccagtgagggcctaaggctcctaatg 1253actttcagggtgagaacaaaaggaattgctctccgccccacccccacctcctgctttccg 1313cagggagacatggaaattcccagttactaaaatagattgtcaatagagttatttatagcc 1373ctcatttcctccggggacttggaagcttcagacagggtttttcataaacaaagtcataac 1433tgatgtgttttacagcatcctagaatcctggcagcctctgaagttctaattaactggaag 1493catttaagcaacacgtcaagtgcccctgctgtggtatttgtttctacttttctgttttta 1553aagtgtgagtcacaaggtaattgttgtaacctgtgatatcactgtttcttgtgtctcttc 1613tttcaactacatcttttaaaacaaaaaaaaaaaaaaaaaaaa 1655

SEQ ID NO:5 is the coding sequence part of SEQ ID NO:3.

The murine B7-DC protein, encoded by the coding region of SEQ ID NO:3,(i.e., by SEQ ID NO:5) has the amino acid sequence SEQ ID NO:4 shownbelow (with leader sequence, transmembrane domain and cytoplasmic tailannotated):

   putative leader sequence 1MLLLLPILNL SLQLHPVAAL FTVTAPKEVY TVDVGSSVSL ECDFDRRECT 50 51ELEGIRASLQ KVENDTSLQS ERATLLEEQL PLGKALFHIP SVQVRDSGQY 100 101RCLVICGAAW DYKYLTVKVK ASYMRIDTRI LEVPGTGEVQ LTCQARGYPL 150 151AEVSWQNVSV PANTSHIRTP EGLYQVTSVL RLKPQPSENT SCMFWNAHMK 200                           putative TM domain 201ELTSAIIDPL SRMEPKVPRT WPLHVFIPAC TTALIFLAIV IIQ RKRI 247                                               cyto. tailThe extracellular domain of this protein is from residue P²⁶ throughresidue W²²¹ The complete DNA sequence of murine B7-DC (originallytermed “butyrophilin-like protein” or “Btdc”) has the Genbank accessionnumber AF142780.2Basic Molecular Approach

This approach is described in more detail in the Examples. The inventorsutilized the PCR Select approach which incorporate two importantfeatures. First, the initial PCR reaction prior to the hybridizationsteps requires only small quantity of RNA. This technique allows the useof highly purified mature DCs that have been rendered substantially freeof contaminating macrophages, progenitor cells or other potentialcontaminating cells. Such highly purified DCs are known to be difficultto obtain in very large numbers. Second, the PCR Select procedureenabled cloning of low copy number, differentially expressed genes.

To identify genes differentially expressed by DCs relative to theircellular counterpart, the activated macrophage, and to identify genesassociated with DC-specific functions, the present inventors applied acDNA subtraction approach. They used a modified PCR-based“representative differential analysis” (RDA) combined with suppressionPCR (PCR Select™) (Diatchenko, L. et al., Proc. Natl. Acad. Sci USA93:66025-6030 (1996)).

General Recombinant DNA Methods

Basic texts disclosing general methods of molecular biology, all ofwhich are incorporated by reference, include: Sambrook, J et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989; Ausubel, F M et al.Current Protocols in Molecular Biology, Vol 2, Wiley-Interscience, NewYork, (current edition); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); Glover, D M, ed, DNA Cloning: A PracticalApproach, vol. I & II, IRL Press, 1985; Albers, B. et al., MolecularBiology of the Cell 2Ed., Garland Publishing, Inc., New York, N.Y.(1989); Watson, J D et al., Recombinant DNA, 2^(nd) Ed, ScientificAmerican Books, New York, 1992; and Old, R W et al., Principles of GeneManipulation: An Introduction to Genetic Engineering, 2^(nd) M Ed.,University of California Press, Berkeley, Calif. (1981).

Unless otherwise indicated, a particular nucleic acid sequence isintended to encompasses conservative substitution variants thereof(e.g., degenerate codon substitutions) and a complementary sequence. Theterm “nucleic acid” is synonymous with “polynucleotide” and is intendedto include a gene, a cDNA molecule, an mRNA molecule, as well as afragment of any of these such as an oligonucleotide, and further,equivalents thereof (explained more fully below). Sizes of nucleic acidsare stated either as kilobases (kb) or base pairs (bp). These areestimates derived from agarose or polyacrylamide gel electrophoresis(PAGE), from nucleic acid sequences which are determined by the user orpublished. Protein size is stated as molecular mass in kilodaltons (kDa)or as length (number of amino acid residues). Protein size is estimatedfrom PAGE, from sequencing, from presumptive amino acid sequences basedon the coding nucleic acid sequence or from published amino acidsequences.

Specifically, cDNA molecules encoding the amino acid sequencecorresponding to B7-DC or fragments or derivatives thereof can besynthesized by the polymerase chain reaction (PCR) (see, for example,U.S. Pat. No. 4,683,202) using primers derived the sequence of theprotein disclosed herein. These cDNA sequences can then be assembledinto a eukaryotic or prokaryotic expression vector and the resultingvector can be used to direct the synthesis of B7-DC, or its fragment orderivative by appropriate host cells, for example COS or CHO cells.

This invention includes isolated nucleic acids having a nucleotidesequence encoding the novel B7-DC, fragments thereof or equivalentsthereof. The term nucleic acid as used herein is intended to includesuch fragments or equivalents. The nucleic acid sequences of thisinvention can be DNA or RNA. A preferred nucleic acid is cDNA encodinghuman B7-DC having the sequence SEQ ID NO:1 or equivalents thereof.

Preferably, the nucleic acid of the present invention is a cDNA moleculeencoding at least a portion of B7-DC. This cDNA can be made from mRNAextracted from mature DCs or other cells naturally expressing thisprotein. A nucleic acid sequence encoding B7-DC is obtainable from DCgenomic DNA. Thus, DNA encoding B7-PC can be cloned from a cDNA or agenomic library in accordance with known protocols.

A cDNA nucleotide sequence encoding B7-DC can be obtained by isolatingtotal mRNA from an appropriate cell line. Double stranded cDNA isprepared from total mRNA. cDNA can be inserted into a suitable plasmid,bacteriophage or viral vector using any one of a number of knowtechniques.

In reference to a nucleotide sequence, the term “equivalent” is intendedto include sequences encoding structurally homologous and/or afunctionally equivalent proteins. For example, a natural polymorphism ofthe B7-DC nucleotide sequence (especially at the third base of a codon)may be manifest as “silent” mutations which do not change the amino acidsequence. However, polymorphisms that involve amino acid sequencechanges in B7-DC may exist in a human (or other mammalian) population.These of skill in the art will appreciate that these allelic variantsthat have changes in one or more nucleotides (up to about 3-4% of thetotal ending sequence) will likely be found in a human population due tonatural allelic variation. Any and all such allelic variations andresulting nucleic acid and polypeptide polymorphisms in the DNA encodingB7-DC are within the scope of the invention.

Furthermore, there may be one or more naturally occurring isoforms orrelated, immunologically cross-reactive family members of the B7-DCprotein described herein. Such isoforms or family members are defined asproteins that share function amino acid sequence similarity to B7-DC,even if they are encoded by genes at different loci.

Fragment of Nucleic Acid

A fragment of the nucleic acid sequence is defined as a nucleotidesequence having fewer nucleotides than the nucleotide sequence encodingthe full length B7-DC protein. This invention includes such nucleic acidfragments that encode polypeptides which retain (1) the ability of B7-DCto bind to its natural ligand(s) on T cells and (2) to enhance orinhibit (depending on how they are presented) activated T cell mediatedimmune responses (measured in cytokine production and/or T cellproliferation by T cells that have received a primary activationsignal).

For example, a nucleic acid fragment as intended herein encodes a B7-DCpolypeptide that retains the ability to bind to the surface of T cellsto a receptor that has not yet been identified (but is does not appearto be CD28 or CTLA-4) and deliver a costimulatory signal to Tlymphocytes. By another criterion, the present nucleic acid fragment isone that hybridizes with a nucleic acid from another animal species andis therefore useful in screening assays to detect novel proteins thatare “cross-reactive” with B7-DC.

Generally, the nucleic acid sequence encoding a fragment of the B7-DCpolypeptide comprises of nucleotides from the sequence encoding themature protein. However, in some instances it may be desirable toinclude all or part of the leader sequence portion of the nucleic acid.Nucleic acid sequences of this invention may also include linkersequences, natural or modified restriction endonuclease sites and othersequences that are useful for manipulations related to cloning,expression or purification of encoded protein or fragments. These andother modifications of nucleic acid sequences are described herein orare well-known in the art.

In one embodiment, DNA encoding the amino acid sequence corresponding tothe ECD of B7-DC, containing amino acids from about position 26-221, isjoined to DNA encoding the amino acid sequences corresponding to thehinge, C_(H)2 and C_(H)3 regions of human Ig Cγ1, using PCR, to form aconstruct that is expressed as B7-DC-Ig fusion protein.

An analogous DNA molecule encoding a B7-Ig fusion protein was disclosedin U.S. Pat. No. 5,521,288 and deposited with the American Type CultureCollection in Rockville, Md., under accession number 68627.

The techniques for assembling and expressing DNA encoding B7-DC andsoluble B7-DC fusion proteins such as synthesis of oligonucleotides,PCR, transforming cells, obstructing vectors, expression systems, andthe like are well-established in the art. Those of ordinary skill aresimilar with the standard resource materials for specific conditions andprocedures.

In other embodiments, the DNA encoding a domain or fragment of B7-DC isfused with a nucleic acid encoding most or all of the remaining portionof another B7 family protein, such as B7.1, B7.2 or B7.3. The completeDNA sequence of human B7.1 (CD80) has the Genbank accession numberX60958; the accession number for the mouse sequence is X60958; theaccession number for the rat sequence is U05593. The complete cDNAsequence of human B7.2 (CD86) has the Genbank accession number L25259;the accession number for the mouse sequence is L25606.

Expression Vectors and Host Cells

This invention includes an expression vector comprising a nucleic acidsequence encoding a B7-DC polypeptide operably linked to at least oneregulatory sequence. “Operably linked” means that the coding sequence islinked to a regulatory sequence in a manner that allows expression ofthe coding sequence. Known regulatory sequences are selected to directexpression of the desired protein in an appropriate host cell.Accordingly, the term “regulatory sequence” includes promoters,enhancers and other expression control elements. Such regulatorysequences are described in, for example, Goeddel, Gene ExpressionTechnology. Methods in Enzymology, vol, 185, Academic Press, San Diego,Calif. (1990)).

Those skilled in the art appreciate that the particular design of anexpression vector of this invention depends on considerations such asthe host cell to be transfected and/or the type of protein to beexpressed.

The present expression vectors comprise the full range of nucleic acidmolecules encoding the various embodiments of B7-DC: full length proteinand its functional derivatives (defined herein) including polypeptidefragments, variants, fusion proteins, etc. Thus, in one embodiment, theexpression vector comprises a nucleic acid encoding at least a portionof the B7-DC protein such as the ECD, alone or fused to another protein.

Such expression vectors are used to transfect host cells for expressionof the DNA and production of the encoded proteins which include fusionproteins or peptides. It will be understood that a genetically modifiedcell expressing the B7-DC polypeptide may transiently express theexogenous DNA for a time sufficient for the cell to be useful for itsstated purpose. Thus, if the cell is to serve as an immunogen having anaugmented costimulatory capacity in vivo or ex vivo, the length of timethat expression is required, or that the cell remain alive, is the timenecessary for the cell to exert its immunogenic and/or costimulatoryfunction. For example, in the case of a transduced tumor cell expressingthe B7-DC of the present invention, expression of B7-DC may be for aslittle as 6 hours, preferably 24 hours, more preferably for at least 2-4days. Of course, expression may also be stable (i.e., for the life ofthe cell). Appropriate expression vectors and regulatory elements (e.g.,(e.g., inducible or constitutive promoters) discussed below are selectedin accordance with the desired or required stability of expression.

The present in invention provides methods for producing the B7-DCprotein, fragments and derivatives. For example, a host cell transfectedwith a nucleic acid vector that encodes at least a portion of the B7-DCprotein is entered under appropriate condition to allow expression ofB7-DC polypeptide.

Host cells may also be transfected with one or more expression vectorsthat singly or in combination comprise DNA encoding at least a portionof the B7-DC protein and DNA encoding at least a portion of a secondprotein, so that the host cells produce fusion polypeptides that includeboth the portions.

When the recombinant expression vector comprises DNA encoding a portionof B7-DC and DNA encoding another protein, such as human IgCγ1, theresulting fusion protein may have altered solubility, binding affinityand/or valency. A B7-DC Ig fusion protein, for example, is preferablysecreted by transfected host cells in cultures and is therefor isolatedfrom the culture medium. Alternatively, if protein is retained in thecytoplasm, the cells are harvested and lysed and the protein isolatedfrom this lysate.

A culture typically includes host cells, appropriate growth media andother byproducts. Suitable culture media are well known in the art.B7-DC protein can be isolated from medium or cell lysates usingconventional techniques for purifying proteins and peptides, includingammonium sulfate precipitation, fractionation column chromatography(e.g. ion exchange, gel filtration, affinity chromatography, etc.)and/or electrophoresis (see generally, “Enzyme Purification and RelatedTechniques”, Methods in Enzymology, 22:233-577 (1971)). Once purified,partially or to homogeneity, the recombinant B7-DC proteins of theinvention can be utilized in pharmaceutical compositions as described inmore detail herein.

Prokaryotic or eukaryotic host cells transformed or transfected toexpress B7-DC or a homologue or functional derivative thereof are withinthe scope of the invention. For example, B7-DC may be expressed inbacterial cells such as E. coli, insect cells (baculovirus), yeast, ormammalian cells such as Chinese hamster ovary cells (CHO) or humancells. Other suitable host cells may be found in Goeddel, (1990) supraor are otherwise known to those skilled in the art.

Expression in eukaryotic cells leads to partial or completeglycosylation and/or formation of relevant inter- or intra-chaindisulfide bonds of the recombinant protein.

Examples of vectors for expression in yeast S. cerevisiae includepYepSec1 (Baldari et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan et al.(1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., (1983)Mol. Cell Biol. 3:2156-2165,) and the pVL series (Lucklow, V. A., andSummers, M. D., (1989) Virology 170: 31-39). Generally, COS cells(Gluzman, Y., (1981) Cell 23: 175-182) are used in conjunction with suchvectors as pCDM 8 (Aruffo A. and Seed, B., supra, for transientamplification/expression in mammalian cells, while CHO (dhfr-negativeCHO) cells are used with vectors such as pMT2PC (Kaufman et al. (1987),EMBO J. 6: 187-195) for stable amplification/expression in mammaliancells. The NS0 myeloma cell line (a glutamine synthetase expressionsystem.) is available from Celltech Ltd.

Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the reporter group and the target proteinto enable separation of the target protein from the reporter groupsubsequent to purification of the fusion protein. Proteolytic enzymesfor such cleavage and their recognition sequences include Factor Xa,thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase,maltose E binding protein, or protein A, respectively, to the targetrecombinant protein.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89). While target gene expression relies on host RNApolymerase transcription from the hybrid trp-lac fusion promoter inpTrc, expression of target genes inserted into pET 11d relies ontranscription from the T7 gn10-lacO fusion promoter mediated bycoexpressed viral RNA polymerase (T7gn1). Th is viral polymerase issupplied by host strains BL21 (DE3) or HMS174 (DE3) from a resident λprophage harboring a T7gn1 under the transcriptional control of thelacUV 5 promoter.

One embodiment of this invention is a transfected cell which expressesnovel B7-DC de novo. In the case of a cell already expressing B7-DC,such as a mature DC, the transfected cell expresses increased amounts ofB7-DC proteins or fragments thereof on the cell surface.

For example, a tumor cell such as a sarcoma, melanoma, leukemia,lymphoma, carcinoma or neuroblastoma is transfected with an expressionvector directing the expression of B7-DC on the tumor cell surface. Suchtransfected tumor cells can be used as immunogens to induce therapeuticantitumor immunity as described herein.

Vector Construction

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and re-ligated inthe form desired.

The DNA sequences which form the vectors are available from a number ofsources. Backbone vectors and control systems are generally found onavailable “host” vectors which are used for the bulk of the sequences inconstruction. For the pertinent coding sequence, initial constructionmay be, and usually is, a matter of retrieving the appropriate sequencesfrom cDNA or genomic DNA libraries. However, once the sequence isdisclosed it is possible to synthesize the entire gene sequence in vitrostarting from the individual nucleotide derivatives. The entire genesequence for genes of sizeable length, e.g., 500-1000 bp may be preparedby synthesizing individual overlapping complementary oligonucleotidesand filling in single stranded nonoverlapping portions using DNApolymerase in the presence of the deoxyribonucleotide triphosphates.This approach has been used successfully in the construction of severalgenes of known sequence. See, for example, Edge, M. D., Nature (1981)292:756; Nambair, K. P., et al., Science (1984) 223:1299; and Jay. E., JBiol Chem (1984) 250:6311.

Synthetic oligonucleotides are prepared by either the phosphotriestermethod as described by references cited above or phosphoramidite methodas described by Beaucage, S. L., and Caruthers, M. H., Tet Lett (1981)22:1859; and Matteucci, M. D., and Caruthers, M. H., J Am Chem Soc(1981) 103:3185 and can be prepared using commercially availableautomated oligonucleotide synthesizers. Kinase treatment of singlestrands prior to annealing or for labeling is achieved using an excess,e.g., about 10 units of polynucleotide kinase to 1 nmole substrate inthe presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol,1-2 mM ATP, 1.7 pmoles γ-³²P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1mM EDTA.

Once the components of the desired vectors are thus available, they canbe excised and ligated using standard restriction and ligationprocedures. Site-specific DNA cleavage is performed by treating with thesuitable restriction enzyme (or enzymes) under conditions which aregenerally understood in the art, and the particulars of which arespecified by the manufacturer of these commercially availablerestriction enzymes. See, e.g., New England Biolabs, Product Catalog. Ingeneral, about 1 mg of plasmid or DNA sentence is cleaved by one unit ofenzyme in about 20 ml of buffer solution; in the examples herein,typically, an excess of restriction enzyme is used to insure completedigestion of the DNA substrate. Incubation times of about one hour totwo hours at about 37° C. are workable, although variations can betolerated. After each incubation, protein is removed by extraction withphenol/chloroform, and may be followed by ether extraction, and thenucleic acid recovered from aqueous fractions by precipitation withethanol. If desired, size separation of the cleaved fragments may beperformed by polyacrylamide gel or agarose gel electrophoresis usingstandard techniques. A general description of size separations is foundin Methods in Enzymology (1980) 65:499-560.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 min at 20° to 25° C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6mM MgCl₂, 6 mM DTT and 0.1-1.0 mM dNTPs. The Klenow fragment fills in at5′ single-stranded overhangs hut chews back protruding 3′ singlestrands, even though the four dNTPs are present. If desired, selectiverepair can be performed by supplying only one of the, or selected, dNTPswithin the limitations dictated by the nature of the overhang. Aftertreatment with Klenow, the mixture is extracted with phenol/chloroformand ethanol precipitated. Treatment under appropriate conditions with S1nuclease or BAL-31 results in hydrolysis of any single-stranded portion.

Ligations are typically performed in 15-50 ml volumes under thefollowing standard conditions and temperatures: for example, 20 mMTris-HCl pH 7.5, 10 mM MgCl₂, 10 mM DTT, 33 μg/ml BSA, 10-50 mM NaCl,and either 40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C.(for “sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNAligase at 14+ C. (for “blunt end” ligation). Intermolecular “sticky end”ligations are usually performed at 33-100 μg/ml total DNA concentrations(5-100 nM total end concentration). Intermolecular blunt end ligationsare performed at 1 mM total ends concentration.

In vector construction employing “vector fragments”, the fragment iscommonly treated with bacterial alkaline phosphatase (BAP) or calfintestinal alkaline phosphatase (CIAP) in order to remove the 5′phosphate and prevent self-ligation. Digestions are conducted at pH 8 inapproximately 10 mM Tris-HCl, 1 mM EDTA using BAP or CIAP at about 1unit/mg vector at 60° C. for about one hour. The preparation isextracted with phenol/chloroform and ethanol precipitated.Alternatively, re-ligation can be prevented in vectors which have beendouble digested by additional restriction enzyme and separation of theunwanted fragments.

Any of a number of methods are used to introduce mutations into thecoding sequence to generate the variants of the invention. Thesemutations include simple deletions or insertions, systematic deletions,insertions or substitutions of clusters of bases or substitutions ofsingle bases.

For example, modifications of B7-DC DNA sequence (cDNA or genomic DNA)are created by site-directed mutagenesis, a well-known technique forwhich protocols and reagents are commercially available (Zoller, M J etal., Nucleic Acids Res (1982) 10:6487-6500 and Adelman, J P et al., DNA(1983) 2:183-193)). Correct ligations for plasmid construction areconfirmed, for example, by first transforming E. coli strain MC1061(Casadaban, M., et al., J Mol Biol (1980) 138:179-207) or other suitablehost with the ligation mixture. Using conventional methods,transformants are selected based on the presence of the ampicillin-,tetracycline- or other antibiotic resistance gene (or other selectablemarker) depending on the mode of plasmid construction. Plasmids are thenprepared from the transformants with optional chloramphenicolamplification optionally following chloramphenicol amplification((Clewell, D B et al., Proc Natl Acad Sci USA (1969) 62:1159; Clewell,D. B., J Bacteriol (1972) 110.667). Several mini DNA preps are commonlyused. See, e.g., Holmes, D S, et al., Anal Biochem (1981) 114:193-197;Birnboim, H C et al., Nucleic Acids Res (1979) 7:1513-1523. The isolatedDNA is analyzed by restriction and/or sequenced by the dideoxynucleotide method of Sanger (Proc Natl Acad Sci USA (1977) 74:5463) asfurther described by Messing, et al., Nucleic Acids Res (1981) 9:309, orby the method of Maxam et al. Methods in Enzymology (1980) 65:499.

Vector DNA can be introduced into mammalian cells via conventionaltechniques such as calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming host cells can befound in Sambrook et al. supra and other standard texts.

Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the reporter group and the target proteinto enable separation of the target protein from the reporter groupsubsequent to purification of the fusion protein. Proteolytic enzymesfor such cleavage and their recognition sequences include Factor Xathrombin and enterokinase.

Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase,maltose E binding protein, or protein A, respectively, to the targetrecombinant protein.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene 69:301-315) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89). While target gene expression relies on host RNApolymerase transcription from the hybrid trp-lac fusion promoter inpTrc, expression of target genes inserted into pET 11d relies ontranscription from the T7 gn10-lacO fusion promoter mediated bycoexpressed viral RNA polymerase (T7gn1). Th is vital polymerase issupplied by host strains BL21(DE3) or HMSI74(DE3) from a resident λprophage harboring a T7gn1 under the transcriptional control of thelacUV 5 promoter.

Promoters and Enhancers

A promoter region of a DNA or RNA molecule binds RNA polymerase andpromotes the transcription of an “operably linked” nucleic acidsequence. As used herein a “promoter sequence” is the nucleotidesequence of the promoter which is found on that strand of the DNA or RNAwhich is transcribed by the RNA polymerase. Two sequences of a nucleicacid molecule, such as a promoter and a coding sequence, are “operablylinked” when they are linked to each other in a manner which permitsboth sequences to be transcribed onto the same RNA transcript or permitsan RNA transcript begun in one sequence to be extended into the secondsequence. Thus, two sequences, such as a promoter sequence and a codingsequence of DNA or RNA are operably linked if transcription commencingin the promoter sequence will produce an RNA transcript of the operablylinked coding sequence. In order to the “operably linked” it is notnecessary that two sequences be immediately adjacent to one another inthe linear sequence.

The preferred promoter sequences of the present invention must beoperable in mammalian cells and may be either eukaryotic or viralpromoters. Suitable promoters may be inducible, repressible orconstitutive. An example of a constitutive promoter is the viralpromoter MSV-LTR, which is efficient and active in a variety of celltypes, and, in contrast to most other promoters, has the same enhancingactivity in arrested and growing cells. Other preferred viral promotersinclude that present in the CMV-LTR (from cytomegalovirus) (Bashart, M.et al., Cell 41-521 (1985)) or in the RSV-LTR (from Rous sarcoma virus)(Gorman, C. M., Proc. Natl. Acad. Sci. USA 79:6777 (1982). Also usefulare the promoter of the mouse metallothionein I gene (Hamer, D., et al.,J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus(McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter(Benoist, C., et al., Nature 290:304-310 (1981)); and the yeast gal4gene promoter (Johnston, S. A., et al., Proc. Natl. Acad. Sci. (USA)79:6971-6975 (1982); Silver, P. A., et al., Proc. Natl. Acad. Sci. (USA)81:5951-5955 (1984)). Other illustrative descriptions of transcriptionalfactor association with promoter regions and the separate activation andDNA binding of transcription factors include: Keegan et al., Nature(1986) 231:699; Fields et al., Nature (1989) 340:245; Jones, Cell (1990)61:9; Lewin, Cell (1990) 61:1161; Ptashne et al., Nature (1990) 346:329;Adams et al., Cell (1993) 72:306. The relevant disclosure of all ofthese above-listed references is hereby incorporated by reference.

The promoter region may further include an octamer region which may alsofunction as a tissue specific enhancer, by interacting with certainproteins found in the specific tissue. The enhancer domain of the DNAconstruct of the present invention is one which is specific for thetarget cells to be transfected, or is highly activated by cellularfactors of such target cells. Examples of vectors (plasmid orretrovirus) are disclosed in (Roy-Burman et al., U.S. Pat. No.5,112,767). For a general discussion of enhancers and their actions intranscription, see, Lewin, B. M., Genes IV, Oxford University Press,Oxford, (1990), pp. 552-576. Particularly useful are retroviralenhancers (e.g., viral LTR). The enhancer is preferably placed upstreamfrom the promoter with which it interacts to stimulate gene expression.For use with retroviral vectors, the endogenous viral LTR may berendered enhancer-less and substituted with other desired enhancersequences which confer tissue specificity or other desirable propertiessuch as transcriptional efficiency on the B7-DC-encoding DNA molecule ofthe present invention.

The nucleic acid sequences of the invention can also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxy-nucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated withcommercially available DNA synthesizers (See, e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated byreference herein).

Proteins and Polypeptides

The present invention includes an “isolated” B7-DC protein having thesequence SEQ ID NO:2 or SEQ ID NO:4. While the present disclosureexemplifies the full length human and murine B7-DC protein (and DNA), itis to be understood that homologues of B7-DC from other mammalianspecies and mutants thereof that possess the characteristics disclosedherein are intended within the scope of this invention.

Also included is a “functional derivative” of B7-DC which is means inamino acid substitution variant, a “fragment,” or a “chemicalderivative” of B7-DC, which terms are defined below. A functionalderivative retains measurable B7-DC activity, preferably that of bindingto a receptor on T cells and costimulating T cell activity, whichpermits its utility in accordance with the present invention.“Functional derivatives” encompass “variants” and “fragments” regardlessof whether the terms are used in the conjunctive or the alternativeherein.

A functional homologue must possess the above biochemical and biologicalactivity. In view of this functional characterization, use of homologousproteins B7-DC from other species, including proteins not yetdiscovered, fall within the scope of the invention if these proteinshave sequence similarity and the recited biochemical and biologicalactivity.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred method of alignment, Cys residues are aligned.

In a preferred embodiment, the length of a sequence being compared is atleast 30%, preferably at least 40%, more preferably at least 50%, evenmore preferably at least 60%, and even more preferably at least 70%,80%, or 90% of the length of the reference sequence. For example, whenaligning a second sequence to the human B7-DC protein amino acidsequence (SEQ ID NO:2) having 276 amino acid residues, at least 83,preferably at least 110, more preferably at least 138, even morepreferably at least 166, and even more preferably at least 193, 221 or248 amino acid residues are aligned). The amino acid residues (ornucleotides) at corresponding amino acid positions (or nucleotide)positions are then compared. When a position in the first sequence isoccupied by the same amino acid residue (or nucleotide) as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences in a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sentences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. 48:444-453 (1970) algorithm which has been incorporated intothe GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases, for example, to identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to human or murine B7-DC nucleic acid molecules. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to human ormurine B7-DC protein molecules of the invention. To obtain gappedalignments for comparison purposes. Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov.

Thus, a homologue of the B7-DC protein described above is characterizedas saving (a) functional activity of native B7-DC, and (b) sequencesimilarity to a native B7-DC protein (such as SEQ ID NO:2 or SEQ IDNO:4, when determined above, of at least about 30% (at the amino acidlevel), preferably at least about 50%, more preferably at least about70%, even more preferably at least about 90%.

It is within the skill in the art to obtain and express such a proteinusing DNA probes based on the disclosed sequences of B7-DC. Then, theprotein's biochemical and biological activity can be tested readilyusing art-recognized methods such as those described herein, forexample, a standard T cell proliferation or cytokines secretion assay. Abiological assay of T cell co-stimulation will indicate whether thehomologue has the requisite activity to qualify as a “functional”homologue.

Preferred assays measure the functional characteristics of B7-DC such asstimulating T cells synthesis of cytokines, which depends on binding orcross-linking of the TCR (“primary activation signal”), as well asdelivery of a costimulatory signal. The binding of B7-DC to its naturalligand(s) on T cells transmits a signal that induces increased cytokineproduction, such as IL-2, which in turn stimulates proliferation whichcan also be measured routinely.

A “variant” of B7-DC refers to a molecule substantially identical toeither the full protein or to a fragment thereof in which one or moreamino acid residues have been replaced (substitution variant) or whichhas one or several residues deleted (deletion variant) or added(addition variant). A “fragment” of B7 -DC refers to any subset of themolecule, preferably one that includes the ECD, that is, a shorterpolypeptide of the full-length length protein.

A number of processes can be used to generate fragments, mutants andvariants, of the isolated DNA sequence. Small subregions or fragments ofthe nucleic acid encoding the B7-DC protein, for example 1-30 bases inlength, can be prepared by standard, chemical synthesis. Antisenseoligonucleotides and primers for use in the generation of largersynthetic fragment.

A preferred functional derivative is a fusion protein, a polypeptidethat includes a functional fragment of B7-DC. For example, a usefulderivative of B7 is a B7-DC-Ig fusion protein that comprises apolypeptide corresponding to the ECD of B7-DC and an Ig C region. Thepresence of the fusion partner can alter the solubility, affinity and/orvalency (defined here as the number of binding sites available permolecule) of the B7-DC protein. A soluble B7-DC fusion protein, whilestill binding to a receptor on T cells, may have a different biologicaleffect than of the native protein expressed on an APC, i.e., inhibitionof T cell stimulation by competitive binding rather than costimulation.

As used herein an extracellular domain (ECD) of B7-DC is the entireextracellular portion of the protein or any fragment thereof thatrecognizes and binds to PD-1 or to another receptor on T cells that isnot CD28 or CTLA-4. Preferably, an ECD of B7-DC is that portion encodedby amino acid residues from about position 26 to about position 221 ofSEQ ID NO:2 or SEQ ID NO:4.

By “soluble B7-DC” is intended a cell-free form of B7-DC that may beshed, secreted or otherwise extracted from the producing cells. SolubleB7-DC includes, but is not limited to, soluble fusion proteins such asB7-DC-Ig, free ECD of B7-DC, or the B7-DC ECD fused (genetically orchemically) to a biologically active molecule.

As indicated earlier, this invention also includes hybrid fusionproteins between a B7-DC domain and a domain or fragment of another B7family protein, preferably expressed on the cell surface incostimulatory form.

A preferred group of B7-DC variants are those in which at least oneamino acid residue and preferably, only one, has been substituted bydifferent residue. For a detailed description of protein chemistry andstructure, see Schulz, G E et al., Principles of Protein Structure,Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,1983, which are hereby incorporated by reference. The types ofsubstitutions that may be made in the protein molecule may be based onanalysis of the frequencies of amino acid changes between a homologousprotein of different species, such as those presented in Table 1-2 ofSchulz et al. (supra) and FIGS. 3-9 of Creighton (supra). Based on suchan analysis, conservative substitutions are defined herein as exchangeswithin one of the following five groups:

1 Small aliphatic, nonpolar or slightly polar Ala, Ser, Thr (Pro, Gly);residues 2 Polar, negatively charged residues and Asp, Asn, Glu, Gln;their amides 3 Polar, positively charged residues His, Arg, Lys; 4 Largealiphatic, nonpolar residues Met, Leu, Ile, Val (Cys) 5 Large aromaticresidues Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking a side chain andthus imparts flexibility to the chain. Pro, because of its unusualgeometry, tightly constrains the chain. Cys can participate in disulfidebond formation, which is important in protein folding.

More substantial changes in biochemical, functional (or immunological)properties are made by selecting substitutions that are lessconservative, such as between, rather than within, the above fivegroups. Such changes will differ more significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Examples of such substitutions are (i)substitution of Gly and/or Pro by another amino acid or deletion orinsertion of Gly or Pro; (ii) substitution of a hydrophilic residue,e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g., Leu, Ile,Phe, Val or Ala; (iii) substitution of a Cys residue for (or by) anyother residue; (iv) substitution of a residue having an electropositiveside chain, e.g., Lys, Arg or His, for (or by) a residue having anelectronegative charge, e.g., Glu or Asp; or (v) substitution of aresidue having a bulky side chain, e.g., Phe, for (or by) a residue nothaving such a side chain, e.g., Gly.

Most acceptable deletions, insertions and substitutions according to thepresent invention are those that do not produce radical changes in thecharacteristics of the B7-DC protein in terms of its T cellcostimulatory activity. However, when it is difficult to predict theexact effect of the substitution, deletion or insertion in advance ofdoing so, one skilled in the art will appreciate that the effect an beevaluated by routine screening assays such as those described here,without requiring undue experimentation.

Whereas shorter chain variants can be made by chemical synthesis, forthe present invention, the preferred longer chain variants are typicallymade by site-specific mutagenesis of th nucleic acid encoding the B7-DCpolypeptide, expression of the variant nucleic acid in cell culture,and, optionally, purification of the polypeptide from the cell culture,for example, by immunoaffinity chromatography using specific antibodyimmobilized to a column (to absorb the variant by binding to at leastone epitope).

Chemical Derivatives of B7-DC

“Chemical derivatives” of B7-DC contain additional chemical moieties notnormally a part of the protein. Covalent modifications of thepolypeptide are included within the scope of this invention. Suchderivatized moieties may improve the solubility, absorption, biologicalhalf life, and the like. Moieties capable of mediating such effects aredisclosed, for example, in Remington's Pharmaceutical Sciences, 16^(th)ed., Mack Publishing Co., Easton, Pa. (1980).

Such modifications may be introduced into the molecule by reactingtargeted amino acid residues of the polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor terminal residues. Another modification is cyclization of theprotein.

Examples of chemical derivatives of the polypeptide follow.

Lysinyl and amino terminal residues are derivatized with succinic orother carboxylic acid anhydrides. Derivatization with a cycle carboxylicanhydride has the effect of reversing the charge of the lysinylresidues. Other suitable reagents for derivatizing amino-containingresidues include imidoesters such as methyl picolinimidate; pyridoxalphosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Carboxyl side groups, aspartyl or glutamyl, may be selectively modifiedby reaction with carbodiimides (R—N═C═N—R′) such, as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonia.

Other modifications include hydroxylation of proline and lysine,phosphorylation, of hydroxyl groups of seryl or threonyl residues,methylation of the amino group of lysine (Creighton, supra, pp. 79-86),acetylation of the N-terminal amine, and amidation of the C-terminalcarboxyl groups.

Also included are peptides wherein one or more D-amino acids aresubstituted for one or more L-amino acids.

Multimeric Peptides

The present invention also includes longer polypeptides in which a basicpeptidic sequence obtained from the sequence of B7-DC is repeated fromabout two to about 100 times, with or without intervening spacers orlinkers. It is understood that such multimers may be built from any ofthe peptide variants defined herein. Moreover, a peptide multimer maycomprise different combinations of peptide monomers and the disclosedsubstitution variants thereof. Such oligomeric or multimeric peptidescan be made by chemical synthesis or by recombinant DNA techniques asdiscussed herein. When produced chemically, the oligomers preferablyhave from 2-8 repeats of the basic peptide sequence. When producedrecombinantly, the multimers may have as many repeats as the expressionsystem permits, for example from two to about 100 repeats.

In tandem multimers, preferably dimers and tamers, of the B7-DC peptideor polypeptide, the chains bonded by interchain disulfide bonds or other“artificial” covalent bonds between the chains such that the chains are“side-by-side” rather than “end to end”. Preferred dimers and trimersare those between fusion proteins of B7-DC such as B7-DC-Ig, asdescribed herein.

Antibodies Specific for Epitopes of B7-DC

In the following description, reference will be made to variousmethodologies known to those of skill in the art of immunology, cellbiology, and molecular biology. Publications and other materials settingforth such known methodologies to which reference is made areincorporated herein by reference in their entireties as though set forthin full. Standard reference works setting forth the general principlesof immunology include A. K. Abbas et al., Cellular and MolecularImmunology (Fourth Ed.), W.B. Saunders Co., Philadelphia, 2000; C. A.Janeway et al., Immunobiology. The Immune System in health and Diseases,Fourth ed., Garland Publishing Co., New York, 1999; Roitt, I. et al.,Immunology, (current ed.) C. V. Mosby Co., St. Louis, Mo. (1999); Klein,J. Immunology, Blackwell Scientific Publication, Inc., Cambridge, Mass.,(1990).

Monoclonal antibodies (mAbs) and methods for their production and useare described in Kohler and Milstein, Nature 256:495-497 (1975); U.S.Pat. No. 4,376,110; Hartlow, E. et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988);Monoclonal Antibodies and Hybridomas: A New Dimension in BiologicalAnalyses, Plenum Press, New York, N.Y. (1980); H. Zola et al., inMonoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,1982)).

Immunoassay methods are also described in Coligan, J. E. et al, eds.,Current Protocols in Immunology, Wiley-Interscience, New York 1991 (orcurrent edition); Butt, W. R. (ed.) Practical Immunoassay: The State ofthe Art, Dekker, New York, 1984; Bizollon, Ch. A., ed., MonoclonalAntibodies and New Trends in Immunoassays, Elsevier, N.Y., 1984; Butler,J. E., ELISA (Chapter 29), in: van Oss, C. J. et al., (eds),IMMUNOCHEMISTRY, Marcel Dekker, inc., New York, 1994, pp. 759-803;Butler, J. E. (ed.), Immunochemistry of Solid-Phase Immunoassay, CRCPress, Boca Raton, 1991; Weintraub, B., Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques, The EndocrineSociety, March, 1986; Work, T. S. et al., Laboratory Techniques andBiochemistry in Molecular Biology, North Holland Publishing Company, NY,(1978) (Chapter by Chard, T., “An introduction to Radioimmune Assay andRelated Techniques”).

Anti-idiotypic antibodies are described, for example, in Idiotypy inBiology and Medicine, Academic Press, New York, 1984; ImmunologicalReviews Volume 79, 1984; Immunological Reviews Volume 90, 1986; Curr.Top. Microbiol., Immunol. Volume 119, 1985; Bona, C. et al., CRC Crit.Rev. Immunol., pp. 33-81 (1981); Jerne, N K, Ann. Immunol. 125C:373-389(1974); Jerne, N K, in: Idiotypes—Antigens on the Inside,Western-Schnurr, I., ed., Editiones Roche, Basel, 1982, Urbain, J etal., Ann. Immunol. 133D:179- (1982); Rajewsky, K. et al., Ann. Rev.Immunol. 1:569-607 (1983)

The present invention provides antibodies, both polyclonal andmonoclonal, reactive with novel epitopes of B7-DC that are absent fromknown B7 family proteins. The antibodies may be xenogeneic, allogeneic,syngeneic, or modified forms thereof, such as humanized or chimericantibodies. Antiidiotypic antibodies specific for the idiotype of ananti-B7-DC antibody are also included. The term “antibody” is also meantto include both intact molecules as well as fragments thereof thatinclude the antigen-binding site and are capable of binding to a B7-DCepitope. These include, Fab and F(ab′)₂ fragments which lack the Fcfragment of an intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Also included are Fvfragments (Hochman, J. et al. (1973) Biochemistry 12:1130-1135; Sharon,J. et al. (1976) Biochemistry 15:1591-1594).). These various fragmentsare be produced using conventional techniques such as protease cleavageor chemical cleavage (see, e.g., Rousseaux et al., Meth. Enzymol.,121:663-69 (1986))

Polyclonal antibodies are obtained as sera from immunized animals suchas rabbits, goats, rodents, etc. and may be used directly withoutfurther treatment or may be subjected to conventional enrichment orpurification methods such as ammonium sulfate precipitation, ionexchange chromatography, and affinity chromatography (see Zola et al.,supra).

The immunogen may comprise the complete B7-D6 protein, or fragments orderivatives thereof. Preferred immunogens comprise all or a part of theECD of human B7-DC (amino acid residues 26-221), where these residuescontain the post-translation modifications, such as glycosylation, foundon the native B7-DC. Immunogens comprising the extracellular domain areproduced in a variety of ways known in the art, e.g., expression ofcloned genes using conventional recombinant methods, isolation fromcells of origin, cell populations expressing high levels of B7-DC, etc.

The mAbs may be produced using conventional hybridoma technology, suchas the procedures introduced by Kohler and Milstein (Nature, 256:495-97(1975)), and modifications thereof (see above references). An animal,preferably a mouse is primed by immunization with an immunogen as aboveto elicit the desired antibody response in the primed animal.

B lymphocytes from the lymph nodes, spleens or peripheral blood of aprimed, animal are fused with myeloma cells, generally in the presenceof a fusion promoting agent such as polyethylene glycol (PEG). Any of anumber of murine myeloma cell lines are available for such use: theP3-NS1/1-Ag4-1, P3-x63-k0Ag8.653, Sp2/0-Ag14, or HL1-653 myeloma lines(available from the ATCC, Rockville, Md.). Subsequent steps includegrowth in selective medium so that unfused parental myeloma cells anddonor lymphocyte cells eventually the while only the hybridoma cellssurvive. These are cloned and grown and their supernatants screened forthe presence of antibody of the desired specificity, e.g. by immunoassaytechniques using the B7-DC-Ig fusion protein Positive clones aresubcloned, e.g., by limiting dilution, and the mAbs are isolated.

Hybridomas produced according to these methods can be propagated invitro or in vivo (in ascites fluid) using techniques known in the art(see generally Fink et al., Prog. Clin. Pathol. 9:121-33 (1984)).Generally, the individual cell line is propagated in culture and theculture medium containing high concentrations of a single mAb can beharvested by decantation, filtration, or centrifugation.

The antibody may be produced as a single chain antibody or scFv insteadof the normal multimeric structure. Single chain antibodies include thehypervariable regions from an Ig of interest and recreate the antigenbinding site of the native Ig while being a fraction of the size of theintact Ig (Skerra, A. et al. (1988) Science, 240: 1038-1041; Pluckthun,A. et al. (1989) Methods Enzymol. 178: 497-515; Winter, G. et al. (1991)i Nature, 349: 293-299); Bird et al., (1988) Science 242:423; Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879; Jost C R et al., J BiolChem. 1994 269:26267-26273; U.S. Pat. Nos. 4,704,692, 4,853,871,4,946,778, 5,260,203, 5,455,0Kn contacted with the solution containingan unknown quantity of labeled antibody (which functions as a “reportermolecule”). After a second incubation period to permit the labeledantibody to complex with the antigen bound to the solid support throughthe unlabeled antibody, the solid support is washed a second time toremove the unreacted labeled antibody. This type of forward sandwichassay may be a simple “yes/no” assay to determine whether antigen ispresent or may be made quantitative by comparing the measure of labeledantibody with that obtained for a standard sample containing knownquantities of antigen.

In another type of “sandwich” assay the so-called “simultaneous” and“reverse” assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support and labeledantibody are both added to the sample being tested at the same time.After the incubation is completed, the solid support is washed to removethe residue of fluid sample and uncomplexed labeled antibody. Thepresence of labeled antibody associated with the solid support is thendetermined as it would be in a conventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support after a suitable incubation period isutilized. After a second incubation, the solid phase is washed inconventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support is then determinedas in the “simultaneous” and “forward” assays.

The foregoing antibodies are useful in method for inhibiting T cellsimulation and treating diseases associated with undesired T cellactivation, such as transplant rejection and autoimmunity. This methodinvolves administering a subject in need of such treatment an effectiveamount of an antibody, preferably a mAb, more preferably a human oorhumanized mAb specific for a costimulatory epitope of B7-DC. Theadministration of antibody must be effective in blocking simulation of Tcells or in eliminating antigen-reactive T cells, thereby inhibiting thetargeted T cell response. Relevant dose ranges are described below.

Uses of Nucleic Acids that Encode B7-DC Protein

The nucleic acids of this invention are used diagnostically to monitorthe progress of a disease, by measuring the expression of B7DC in cellsfrom biological samples or for assaying the effect of an agent on theexpression of B7-DC. This is preferably accomplished by measurement ofcellular mRNA levels. For use in such diagnostic methods, the nucleicacid sequence is delectably labeled, e.g., with a radioactive orfluorescent label biotin and used in a conventional dot blot or Northernhybridization procedure to probe mRNA molecules present in, for example,a preparation of total kor poly(A+) RNA from a biological sample.

Therapeutic Compositions and their Administration

The B7-DC polypeptide or a cell expressing this polypeptide such as a DCor a tumor cell is administered to a mammalian subject, preferably ahuman. Cell-associated, immobilized or otherwise aggregated forms of thepolypeptide are used to enhance T lymphocyte reactivity and theresultant immunity. The B7-DC-Ig fusion protein assembles as a dimerand, as shown in the examples, co-stimulates T cells. Soluble monomericforms of the B7-DC polypeptide can bind to the receptor on T cellswithout stimulating activity and can therefore be considered competitiveinhibitors or antagonists of T cell co-stimulation by a stimulatory formof the molecule. Binding of a such a B7-DC antagonist may suppressongoing T cell reactivity or may interfere with the effect of acostimulatory signal presented by endogenous B7-DC or even by other B7family members acting via their receptors (e.g., CD28 or CTLA-4).

A composition living the activity of B7-DC in described herein isadministered in a pharmaceutically acceptable carrier to a biologicallyeffective or a therapeutically effective amount. The B7-DC polypeptide(or cell expressing the polypeptide) may be given alone or incombination with another protein or peptide such as one having theactivity of another member of the B7 family or another immunostimulatorymolecule Treatment may include administration of an adjuvant, used inits broadest sense in include any nonspecific Immune stimulatingcompound such as interferon. Adjuvants contemplated herein includeresorcinols, non-ionic surfactants such as polyoxyethylene oleyl etherand n-hexadecyl polyethylene ether.

The following doses and amounts also pertain to the antibodies of theinvention when administered to a subject.

A therapeutically effective amount is a dosage that, when given for aneffective period of time, achieves the desired immunological or clinicaleffect.

A therapeutically active amount of a polypeptide having B7-DC activity(or an anti-B7-DC antibody) may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the peptide to elicit a desired response in the individual. Dosageregimes may be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation. A therapeutically effective amounts of theprotein, in cell associated form may be stated in terms of the proteinor cell equivalents.

Thus an effective amount is between about 1 ng and about 1 gram perkilogram of body weight of the recipient, more preferably between about1 μg and 100 mg/kg, more preferably, between about 100 μg and about 100mg/kg. Dosage forms suitable for internal administration preferablycontain (for the latter dose range) from about 0.1 mg to 500 mg ofactive ingredient per unit. The active ingredient may vary from 0.5 to95% by weight based on the total weight of the composition.Alternatively, an effective dose of cells expressing B7-DC, suchpreferably transduced cells such as DC's or inactivated tumor cells, isbetween about 10⁴ and 10⁹ cells, more preferably between about 10⁶ and10⁸ cells per subject, preferably in split doses. Those skilled in theart of immunotherapy will be able to adjust these doses without undueexperimentation.

The active compound B7-DC polypeptide or cell transduced with B7-DC DNA)may be administered in a convergent manner, e.g., injection by aconvenient and effective route. Preferred routes include subcutaneous,intradermal, intravenous and intramuscular routs. Other possible routesinclude oral administration, intrathecal, inhalation, transdermalapplication, or rectal administration. For the treatment of tumors whichhave not been completely resected, direct intratumoral injection is alsointended.

Depending on the route of administration, the active compound may becoated in a material to protect the compound from the action of enzymes,acids and other natural conditions which may activate the compound.Thus, to a administer a polypeptide or peptide having B7-DC activity byan enteral route, it may be necessary to coat the composition with, orco-administer the composition with, a material to prevent itsinactivation. For example, a peptide may be administered to anindividual in an appropriate carrier, diluent or adjuvant,co-administered with enzyme inhibitors (e.g., pancreatic trypsininhibitor, diisopropylfluorophosphate (DEP) and trasylol) or in anappropriate carrier such as liposomes (including water-in-oil-in-wateremulsions as well as conventional liposomes (Strejan et al., (1984) J.Neuroimmunol 7:27).

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

Preferred pharmaceutically acceptable diluents include saline andaqueous buffer solutions. Pharmaceutical compositions suitable forinjection include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. Isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, sodiumchloride may be included in the pharmaceutical compositions. In allcases, the composition should be sterile and should be fluid. It shouldbe stable under the conditions of manufacture and storage and mustinclude preservatives that prevent contamination with microorganismssuch as bacteria and fungi. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for sample, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Parenteral compositions are preferably formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit formrefers to physically discrete units suited as unitary dosages for amammalian subject; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effects to be achieved, and (b) theimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For long instillation, aerosolized solutions are used. In a sprayableaerosol preparations, the active protein may be in combination with asolid or liquid inert carrier material. This may also be packaged in asqueeze bottle or in admixture with a pressurized volatile, normallygaseous propellant. The aerosol preparations can contain solvents,buffers, surfactants, and antioxidants in addition to the protein of theinvention.

For topical application, the proteins of the present invention may beincorporated into topically applied vehicles such as salvos orointments, which have both a soothing effect on the skin as well as ameans for administering the active ingredient directly to the affectedarea.

The carrier for the active ingredient may be either in sprayable ornonsprayable form. Non-sprayable forms can be semi-solid or solid formscomprising a carrier indigenous to topical application and having adynamic viscosity preferably greater than that of water. Suitableformulations include, but are not limited to, solution, suspensions,emulsions, creams, ointments, powders, liniments, salves, and the like.If desired, these may be sterilized or mixed, with auxiliary agents,e.g., preservatives, stabilizers, wetting agents, buffers, or salts forinfluencing osmotic pressure and the like. Examples of preferredvehicles for non-sprayable topical preparations include ointment bases,e.g., polyethylene glycol-1000 (PEG-1000); conventional creams such asHEB cream; gels; as well as petroleum jelly and the like.

Other pharmaceutically acceptable carriers for the B7-DC polypeptideaccording to the present invention are liposomes, pharmaceuticalcompositions in which the active protein is contained either dispersedor variously present in corpuscles consisting of aqueous concentriclayers adherent to lipidic layers. The active protein is preferablypresent in the aqueous layer and in the lipidic layer, inside oroutside, or, in any event, in the non-homogeneous system generally knownas a liposomic suspension. The hydrophobic layer, or lipidic layer,generally, but not exclusively, comprises phospholipids such as lecithinand sphingomyelin, steroids such as cholesterol, more or less ionicsurface active substances such as dicetylphosphate, stearylamine orphosphatidic acid, and/or other materials of a hydrophobic nature.

Modification of Tumor Cells to Express B7-DC and Multiple CostimulatoryMolecules

Another aspect of the invention is a cell, preferably a tumor cellmodified to express multiple costimulatory molecules. The temporalexpression of costimulatory molecules on activated B cells is differentfor B7, B7-2 and B7-3. For example, B7-2 is expressed early following Bcell activation, whereas B7-3 is expressed, later. The differentcostimulatory molecules may thus serve distinct functions during thecourse of an immune response. An effective T cell response may requirethat the T cell receive costimulatory signals from multiplecostimulatory molecules.

Accordingly, the invention encompasses a tumor cell which is geneticallymodified or to express more than one costimulatory molecule. Forexample, a tumor cell can be modified to express B7-DC and one or moreof B7, B7-2 and B7-3.

Before modification, a cell such as a tumor cell may not express anycostimulatory molecules, or may express certain costimulatory moleculesbut not others. As described herein, tumor cells can be modified bytransfection with nucleic acid encoding B7-DC alone or with anothercostimulatory molecule(s). For example, a tumor cell transfected withnucleic acid encoding B7-DC can be further transfected with nucleic acidencoding B7. The sequence of cDNA molecules encoding human or mouseB7-DC proteins are SEQ ID NO:1and the coding portion of SEQ ID NO:3,respectively. Alternatively, more than one type of modification can beused. For example, a tumor cell transfected with a nucleic acid encodingB7-DC can be stimulated with an agent which induces expression of B7-1,B7-2 or B7-3.

Antigens Associated with Pathogens

A major utility for the present invention is the use of the presentcompositions in therapeutic vaccine for cancer and for major chronicviral infections that cause morbidity and mortality worldwide. Suchvaccines are designed to eliminate infected cells—this requires T cellresponses as antibodies are ineffective. The vaccines of the presentinvention, include, in addition to the antigenic epitope itself:

-   (a) a vector such as naked DNA, naked RNA, self replicating RNA    replicons and viruses including vaccinia, adenoviruses,    adeno-associaged virus (AAV), lentiviruses and DNA alphaviruses;-   (b) an antigen targeting or processing signal seen as HSP70,    calreticulin, the extracellular domain of Flt-3 ligand, domain II of    Pseudomonas exotoxin ETA, herpes simplex VP22 targeting protein, and    the like. (See commonly assigned U.S. patent application Ser. Nos.    09/421,608; 09/501/097; 09/693,450; 60/222,9002; 60/222,985;    60/268,575 and chang, W-F et al., J. Virol. 75:2368-2376 (2001),    which are hereby incorporated by reference in their entirety); and-   (c) a costimulatory signal, preferably the B7-DC protein of the    present invention or a fusion protein, fragment or functional    derivative thereof (alone or in combination with other known    costimulatory proteins such as B7.1, B7.2, soluble CD40, etc.).

Tumor cells or other types of host cells, including APCs, aretransformed, transfected or otherwise transduced with a nucleic encodingan antigen to which an immune response is desired. Such antigens arepreferably epitopes of pathogenic microorganisms against which the hostis defended by effector T cells responses, including cytotoxic Tlymphocyte (CTL) and delayed type hypersensitivity. These typicallyinclude viruses, intracellular parasites such as malaria, and bacteria,that grow intracellularly such as mycobacteria and listeria. Thus, thetypes of antigens included in the vaccine compositions of this inventionare any of those associated with such pathogens (in addition, of course,to tumor-specific antigens). It is noteworthy that some viral antigensare also tumor antigens in the case where the virus is a causativefactor in cancer.

In fact, the two most common cancers worldwide, hepatoma and cervicalcancer, are associated with viral infection. Hepatitis B virus (HBV)(Beasley, R. P. et al. Lancet 2, 1129-1133 has been implicated asetiologic agent of hepatomas. 80-90% of cervical cancers express the E6and E7 antigens from one of four “high risk” human papillomavirus types:HPV-16, HPV-18, HPV-31 and HPV-45 (Gissmann, L. et al., Ciba Found Symp.120, 190-207 (1986); Beaudenon, S., et al. Nature 321, 246-249 (1986).The HPV E6 and E7 antigens are the most promising targets for virusassociated cancers in immunocompetent individuals because of theirubiquitous expression in cervical cancer. In addition to theirimportance as targets for therapeutic cancer vaccines, virus associatedtumor antigens are also ideal candidates for prophylactic vaccines.Indeed, introduction of prophylactic HBV vaccines in Asia have decreasedthe incidence of hepatoma (Chang, M. H., et al. New Engl. J. Med. 336,1855-1859 (1997), representing a great impact on cancer prevention.

Among the most important viruses in chronic human viral infections arehuman papillomavirus (HPV) hepatitis B virus (HBV), hepatitis C Virus(HCV), human immunodeficiency virus (HIV), Epstein Barr Virus (EBV) andherpes simplex virus (HSV).

In addition to its applicability to human cancer and current informationdiseases, the present invention is also intended for use in treatinganimal diseases in the veterinary medicine context. Thus, the approachesdescribed herein may be readily applied by one skilled in the art totreatment of veterinary herpesvirus infections including equineherpesviruses, bovine herpesviruses, Marek's disease virus in chickensand other fowl; animal retroviral diseases; pseudorabies and rabies andthe like.

The following references set forth principles and current information inthe field of basic, medical and veterinary virology and are incorporatedby reference: Fields Virology, Fields, B N et al., eds., LippincottWilliams & Wilkins, NY, 1996; Principles of Virology: Molecular Biology,Pathogenesis, and Control, Flint, S. J. et al., eds., Amer Society forMicrobiology, Washington, 1999; Principles and Practice of ClinicalVirology, 4th Edition, Zuckerman, A. J. et al., eds., John Wiley & Sons,NY, 1999; The Hepatitis C Viruses, by Hagedorn, C H et al., eds.,Springer Verlag, 1999; Hepatitis B Virus: Molecular Mechanisms inDisease and Novel Strategies for Therapy, Koshy, R. et al., eds., WorldScientific Pub Co, 1998; Veterinary Virology, Murphy, F. A. et al.,eds., Academic Press, NY, 1999; Avian Viruses: Function and Control,Ritchie, B. W., Iowa State University Press, Ames, 2000; Virus Taxonomy:Classification and Nomenclature of Viruses: Seventh Report of theInternational Committee on Taxonomy of Viruses, by M. H. V. VanRegenmortel, M H V et al., eds., Academic press; NY, 2000.

Targeting Molecules

A number of proteins that have various modes of action have beenimplicated as “targeting” molecules to be used in conjunction withantigens, preferably as fusion polypeptides, to target the antigen tocells and subcellular compartments that promote presentation of theantigen to T cells in a more potent and effective manner.

Linkage of antigens to heat shock proteins (HSPs) represents a potentialapproach for increasing the potency of nucleic acid-based (and other)vaccines. HSPs appear to acct as natural biologic adjuvants in cancerand viral vaccination. Both, the gp96 HSP resident in the endoplasmicreticulum (ER) and the cytosolic Hsp70 act as immunologic adjuvants(Srivastava, P K et al., Semin. Immunol 3, 57-64 (1991); Udono, H etal., Proc. Natl. Acad. Sci. USA 91, 3077-3081 (1994). These HSPs, orchaperonins, bind a wide array of peptides (Lammert, E., et al. Eur. J.Immunol. 27, 923-927 (1997)). Hsp70 is a chaperonin that can targetassociated proteins to the proteosome—the primary cellular proteasecomplex that generates peptides for association with MHC class Imolecules. Therefore, antigens directly linked to Hsp70 are moreefficiently presented by MHC class I (leading, inter alia, to CTLresponses). Two features appear to be responsible for HSP adjuvanticity:(1) in vitro, peptide loaded gp96 effectively introduce antigens intothe MHC class I processing pathway; (2) binding of gp96 to macrophagesinduces secretion of proinflammatory cytokines, thus augmenting thefunction of the cells to which the peptide antigen has been targeted.

Immunization with HSP complexes isolated from tumor or fromvirus-infected cells induces potent antitumor immunity (Srivastava, P Ket al., Int J Cancer. 33: 417-22, 1984; Srivastava, P K et al., ProcNatl Acad Sci USA. 83: 3407-11, 1986; Udono, H et al., J Immunol. 152:5398-5403, 1994; Blachere, N E et al., J Immunother. 14:352-6, 1993;Udono, H et al., supra; Tamura, Y et al., Science, 278; 117-20, 1997;Janetzki, S et al, J Immunother. 21: 269-76, 1998)) or antiviralimmunity (Heikema, A et al., Immunol Lett. 57: 69-74, 1997; Suto, R etal., Science. 269: 1585-8, 1995). Mixing peptides with HSPs in vitrogenerated immunogenic HSP-peptide complexes (Ciupitu, A M et al., J ExpMed. 187:685-91, 1998; Blachere, N E et al., J Exp Med. 186: 1315-22,1997). Some HSP-based protein vaccines involved fusion of the antigen tothe HSP (Suzue, K et al., J Immunol. 156: 873-9, 1996; Suzue, K. et al.,Proc Natl Acad Sci USA 94; 13146-51, 1997). More recently, the presentinventors and their colleagues (e.g., Chen, C-H et al., Canc. Res.60:1035-1042 (2000)) used HSPs in the form of chimeric DNA in RNAreplicon vaccines. They used HPV-16 E7 as antigen, fused toMycobacterium tuberculosis HSP70 and showed increased expansion andactivation of E7-specific CD8+ T cells which resulted in potentantitumor immunity against established tumors (Lin, K.-Y. et al., CancerRes. 56; 21-26., 1996).

Another useful targeting molecule is the translocation domain of aPseudomonas exotoxin A (ETA), e.g., domain II (dII) of ETA (spanningresidues 253-364). A translocation domain is a polypeptide that inducestranslocation of protein or polypeptide to which it is linked into thecytosol of a cell. For example, similarly applicable polypeptide arederived from a Diphtheria, Clostridia botulinum, tetani), Anthrax,Yersinia, Vibrio cholerae, or Bordetella pertussis toxin. The toxicdomain of the DNA encoding the toxin is preferably mutated or deleted inthe preparation of such compositions.

Calreticulun (CRT) is in abundant 46 kDa protein located in theendoplasmic reticulum (ER) lumen that displays lectin activity and isknown to be involved in the folding and assembly of nascentglycoproteins (Hash (1994) Mol. Cell. Biochem. 135:71-78; Hebert (1997)J. Cell Biol. 139:613-623; Vassilakos (1998) Biochemistry 37:3480-3490;Spiro (1996) J. Biol. Chem. 271:1158-11594. CRT associates with peptidestransported into the ER by transporters associated with antigenprocessing, such as TAP-1 and TAP-2 (Spee (1997) Eur. J. Immunol.27:2441-2449). CRT forms complexes in vitro with peptides. Thesecomplexes, when administered to mice, elicited peptide-specific CD8+ Tcell responses (Basu (1999) J. Exp. Med. 189: 797-802; Nair (1999) J.Immunol. 162:6426-6432). CRT purified from mouse tumors elicitedimmunity specific to the tumor used as the source of CRT, but not to anantigenically distinct tumor (Basu, supra). By pulsing DCs in vitro witha CRT bound to a peptide, the peptide was re-presented in the context ofDC Class I molecules and stimulated peptide-specific CTLs (Nair, supra).

The Flt-3 ligand stimulates growth of DC precursors and can promotegeneration of large numbers of DCs in vivo (Maraskovsky, E. et al., JExp Med 184: 1953-62, 1996; Shurin, M R. et al., Cell Immunol.179:174-84, 1997). Flt3, a murine tyrosine kinase receptor (Rosent, O.et al., Oncogene 6: 1641-50, 1991) is a member of the III receptorkinase family (for review see Lyman, S D, Curr Opin Hematol. 5: 192-6,1998). In hematopoietic tissues, the expression of Flt3 is restricted tothe CD34 -positive progenitors. FIt3 was used to identify andsubsequently clone the corresponding ligand, Flt3-ligand (Lyman, S D etal., Cell 75: 1157-67, 1993; Hannum, C et al., Nature 368: 643-8, 1994).The predominant form of Flt3-ligand is synthesized as a transmembraneprotein from which the functionally similar soluble ECD is generated byproteolytic cleavage (Lyman et al., supra). These proteins bind to andactivating unique tyrosine kinase receptors. Among hematopoietic cells,expression of the Flt3 receptor is primarily restricted to the mostprimitive progenitor cells, including DC precursors. The ECD ofFlt3-ligand generated strong anti-tumor effects against several murinemodel tumors including, fibrosarcoma, breast cancer, liver cancer, lungcancer, melanoma and lymphoma (Lynch, D H et al., Nat Med. 3; 625-631,1997; Chen, K. et al., Cancer Res. S7:3511-3516, 1997; Braun, S E etal., Hum Gene Ther. 10: 2141-2151, 1999; Peron, J M et al., J Immunol.161: 6164-6170, 1998; Chakravarty, P K et al., Cancer Res. 59:6028-6032, 1999; Esche, C et al., Cancer Res. 58: 380-383, 1998.) (19).The present inventors' colleagues linked DNA encoding HPV Ek7 protein toDNA encoding Flt3-ligand ECD. Immunization with this constructdramatically increased expansion and activation of E7 antigen-specificCD8⁺ T cells, resulting in potent anti-tumor immunity againstestablished E7-expressing metastatic tumors.

The HSV-1 protein VP22 is a prototype protein that contributes, amongother things, to enhanced spread of antigen due to its remarkableproperty of intercellular transport (Elliott, G., and P. O'Hare. 1997,Cell 88:223-33) can be used. For example, VP22 linked to p53 (Phelan, A.et al, 1998, Nat Biotechnol 16:440-443) or thymidine kinase (Dilber, M Set al., 1999, Gene Ther 6: 12-21), facilitated the spread of linkedprotein to surrounding cells in vitro and the treatment of model tumors.VP22 linked to HPV-16 E7 antigen in the context of a DNA vaccine led toa dramatic increase in the number of B7-specific CD8⁺ T cell precursorsin vaccinated mice (around 50-fold) and converted a loss effective DNAvaccine into one with significant potency against E7-expressing tumors.Anon-spreading VP22 mutant failed to enhance vaccine potency. VP22 andproteins that may have a similar mode of action, contribute in severalways to enhanced vaccine potency: (1) facilitate spreading of antigenfrom transfected cells to surrounding APCs, thereby increasing thenumber of APCs that present antigen through MHC class I pathway; (2)present antigen more efficiently in transfected cells (3) carryout“cross-priming” whereby release of a VP22 /antigen fusion protein leadsto uptake and processing by DCs (or other APCs) for presentation via theMHC-I restricted pathway (Huang, A Y et al., 1994, Science 264:961-965)

Those skilled in the art will know how to identify appropriate epitopes,e.g., CTL epitopes, of the relevant proteins from the pathogens for usein accordance with this invention.

Delivery of B7-DC DNA to Cells and Animals

DNA delivery, for example to effect what is generally known as “genetherapy” involves introduction of a “foreign” DNA into a cell andultimately, into a live animal. Several general strategies for genetherapy have been studied and have been reviewed extensively (Yang,N-S., Crit. Rev. Biotechnol. 12:335-356 (1992); Anderson, W. F., Science256:808-813 (1992); Miller, A. S., Nature 357:455-460 (1992); Crystal,R. G., Amer. J. Med. 92 (suppl 6A):44S-52S (1992); Zwiebel, J. A. etal., Ann. N.Y. Acad. Sci. 618:394-404 (1991); McLachlin, J. R. et al.,Prog. Nucl. Acid Res. Molec. Biol. 38:91-135 (1990); Kohn, D. B. et al.,Cancer Invest 7:179-192 (1989), which references are herein incorporatedby reference in their entirety).

One approach comprises nucleic acid transfer into primary cells inculture followed by autologous transplantation of the ex vivotransformed cells into the host, either systemically or into aparticular organ or tissue.

For accomplishing the objectives of the present invention, nucleic acidtherapy would be accomplished by direct transfer of a the functionallyactive DNA into mammalian somatic tissue or organ in vivo. DNA transfercan be achieved using a number of approaches described below. Thesesystems can be tested for successful expression in vitro by use of aselectable marker (e.g., G418 resistance) to select transfected clonesexpressing the DNA, followed by detection of the presence of the B7-DCexpression product (after treatment with the inducer in the case of aninducible system) using an antibody to the product in an appropriateimmunoassay. Efficiency of the procedure, including DNA uptake, plasmidintegration and stability of integrated plasmids, can be improved bylinearizing the plasmid DNA using known methods, and co-transfectionusing high molecular weight mammalian DNA as a “carrier”.

Examples of successful “gene transfer” reported in the art include; (a)direct injection of plasmid DNA into mouse muscle tissues, which led toexpression of marker genes for an indefinite period of time (Wolff, J.A. et al., Science 247:1465 (1990); Acsadi, G. et al., The New Biologist3:71 (1991)); (b) retroviral vectors are effective for in vivo and insitu infection of blood vessel tissues; (c) portal vein injection anddirect injection of retrovirus preparations into liver effected genetransfer and expression in vivo (Horzaglou, M. et al., J. Biol Chem.265:17285 (1990); Koleko, M. et al., Human Gene Therapy 2:27 (1991);Ferry, N. et al., Proc. Natl. Acad. Sci. USA 88:8387 (1991)); (d)intratracheal infusion of recombinant adenovirus into lung tissues waseffective for in vivo transfer and prolonged expression of foreign genesin lung respiratory epithelium (Rosenfeld, M. A. et al., Science 252:431(1991); (e) Herpes simplex virus vectors achieved in vivo gene transferinto brain tissue (Ahmad, F. et al., eds, Miami Short Reports—Advancesin Gene Technology: The Molecular Biology of Human Genetic Disease, Vol.1, Boerringer Manneheim Biochemicals, USA, 1991).

Retroviral-mediated human therapy utilizes amphotropic,replication-deficient retrovirus systems (Temin, H. M. Human GeneTherapy 1:111 (1990); Temin et al., U.S. Pat. No. 4,980,289; Temin etal., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 5,124,263;Wills, J. W. U.S. Pat. No. 5,175,099; Miller, A. D., U.S. Pat. No.4,861,719). Such vectors have been used to introduce functional DNA intohuman cells or tissues, for example, the adenosine deaminase gene intolymphocytes, the NPT-II gene and the gene for tumor necrosis factor intotumor infiltrating lymphocytes. Retrovirus-mediated gene deliverygenerally requires target cell proliferation for gene transfer (Miller,D. G. et al., Mol. Cell. Biol. 10:4239 (1990). This condition is met bycertain of the preferred target cells into which the present DNAmolecules are to be introduced, i.e., actively growing tumor cells. Genetherapy of cystic fibrosis using transfection by plasmids using any of anumber of methods and by retroviral vectors has been described byCollins et al., U.S. Pat. No. 5,240,846.

The DNA molecules encoding the B7-DC sequences may be packaged intoretrovirus vectors using packaging cell lines that producereplication-defective retroviruses, as is well-known in the art (see,for example, Cone, R. D. et al., Proc. Natl. Acad. Sci. USA 81:6349-6353(1984); Mann, R. F. et al., Cell 33:153-159 (1983); Miller, A. D. etal., Molec. Cell. Biol. 5:431-437 (1985),; Sorge, J., et al., Molec.Cell. Biol. 4:1730-1737 (1984); Hock, R. A. et al., Nature 320:257(1986); Miller, A. D. et al., Molec. Cell. Biol. 6:2895-2902 (1986).Newer packaging cell lines which are efficient an safe for gene transferhave also been described (Bank et al., U.S. Pat. No. 5,278,056.

This approach can be utilized in a site specific manner to deliver theretroviral vector to the tissue or organ of choice. Thus, for example, acatheter delivery system can be used (Nabel, B G et al., Science244:1342 (1989)). Such methods, using either a retroviral vector or aliposome vector, are particularly useful to deliver the nucleic acid tobe expressed to a blood vessel wall, or into the blood circulation of atumor.

Other virus vectors may also be used, including recombinant adenoviruses(Horowitz, M. S., In: Virology, Fields, B N et al., eds, Raven Press,New York, 1990, p. 1679; Berkner, K. L., Biotechniques 6:616 9191988),Strauss, S. E., In: The Adenoviruses, Ginsberg, H S, ed., Plenum Press,New York, 1984, chapter 11),herpes simplex virus (HSV) forneuron-specific delivery and persistence. Advantages of adenovirusvectors for human gene therapy include the fact that recombination israre, no human malignancies are known to be associated with suchviruses, the adenovirus genome is double stranded DNA which can bemanipulated to accept foreign genes of up to 7.5 kb in size, and liveadenovirus is a safe human vaccine organisms. Adeno-associated virus isalso useful for human therapy (Samulski, R. J. et al., EMBO J. 10:3941(1991) according to the present invention.

Another vector which can express the DNA molecule of the presentinvention, and is useful in the present therapeutic setting,particularly in humans, is vaccinia virus, which can be renderednon-replicating (U.S. Pat. Nos. 5,225,336; 5,204,243; 5,155,020;4,769,330; Sutter, G. et al., Proc. Natl. Acad. Sci. USA (1992)89:10847-10851; Fuerst, T. R. et al., Proc. Natl. Acad. Sci. USA (1989)86:2549-2553; Faulkner F. G. et al.; Nucl. Acids Res (1987) 15:7192;Chakrabarti, S et al., Molec. Cell. Biol. (1985) 5:3403-3409).Descriptions of recombinant vaccinia viruses and other virusescontaining heterologous DNA and their uses in immunization and DNAtherapy are reviewed in: Moss, B., Curr. Opin. Genet. Dev. (1993)3:86-90; Moss, B. Biotechnology (1992) 20: 345-362; Moss, B., Curr TopMicrobiol Immunol (1992) 158:25-38; Moss, B. Science (1991)252:1662-1667; Piccini, A et al., Adv. Virus Res. (1988) 34:43-64; Moss,B. et al., Gene Amplif Anal (1993) 3:201-213.

In addition to naked DNA or RNA, or viral vectors, engineered bacteriamay be used as vectors. A number of bacterial strains includingSalmonella, BCG and Listeria monocytogenes (LM) (Hoiseth & Stocker,Nature 291, 238-239 (1981); Poirier, T P et al. J. Exp. Med. 168, 25-32(1988); (Sadoff, J. C., et al., Science 240, 336-338 (1988); Stover, C.K., et al., Nature 351, 456-460 (1991); Aldovini, A. et al., Nature 351,479-482 (1991); Schafer, R., et al., J. Immunol. 149, 53-59 (1992);Ikonomidis, G. et al., J. Exp. Med. 180, 2209-2218 (1994)). Theseorganisms display two promising characteristics for use as vaccinevectors; (1) enteric routes of infection, providing the possibility oforal vaccine delivery; and (2) infection of monocytes/macrophagesthereby targeting antigens to professional APCs.

In addition to virus-mediated gene transfer in vivo, physical meanswell-known in the art can be used for direct transfer of DNA, includingadministration of plasmid DNA (Wolff et al., 1990, supra) andparticle-bombardment mediated gene transfer (Yang, N.-S., et al., Proc.Natl. Acad. Sci. USA 87:9568 mm (1990); Williams, R. S. et al., Proc.Natl. Acad. Sci. USA 88:2726 (1991); Zelenin, A. V., et al., FEBS Lett.280:94 (1991); Zelenin, A.V. et al., FEBS Lett. 244:65 (1989); Johnston,S. A. et al., In Vitro Cell. Dev. Biol. 27:11 (1991)). Furthermore,electroporation, a well-known means to transfer genes into cell invitro, can be used to transfer DNA molecules according to the presentinvention to tissues in vivo (Titomirov, A. V. et al., Biochim. Biophys.Acta 1088:131 ((1991)).

“Carrier mediated gene transfer” has also been described (Wu, C. H. etal., J. Biol. Chem. 264:16985 (1989); Wu, G. Y. et al., J. Biol. Chem.263:14621 (1988); Soriano, P. et al., Proc. Natl. Acad. Sci. USA 80:7128(1983); Wang, C-Y. et al., Proc. Natl. Acad. Sci. USA 84:7851 (1982);Wilson, J. M. et al., J. Biol. Chem, 267:963 (1992)). Preferred carriersare targeted liposomes (Nicolau, C. et al., Proc. Natl. Acad. Sci. USA80:1068 (1983); Soriano et al., supra) such as immunoliposomes, whichcan incorporate acylated mAbs into the lipid bilayer (Wang et al.,supra). Polycations such as asialoglycoprotein/polylysine (Wu et al.,1989, supra) may be used, where the conjugate includes a molecule whichrecognizes the target tissue (e.g., asialoorosomucoid for liver) and aDNA binding compound to bind to the DNA to be transfected. Polylysine isan example of a DNA binding molecule which binds DNA without damagingit. This conjugate is then complexed with plasmid DNA according to thepresent invention for transfer.

Plasmid DNA used for transfection or microinjection may be preparedusing methods well-known in the art, for example using the Quiagenprocedure (Quiagen), followed by DNA purification using known methods,such as the methods exemplified herein.

Again, as noted above, for the utility of transduced B7-DC moleculesaccording to this invention may not require stable expression. Rather,transient expression of the polypeptide may be sufficient for transducedcells to perform their immunogenic and/or costimulatory function.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE I Materials and Methods

Cell Preparation and Culture

6-12 week old female BALB/c mice were purchased from NCI and used for DCand macrophage preparation.

Bone marrow-derived DCs were cultured in RPMI1640 (Gibco BRL) mediumsupplemented with 5% fetal calf serum (FCS) (Hyclone),Penicillin/Streptomycin (JRH Biosciences), Gentamycin (Sigma),Nonessential amino acids (JRH Biosciences), L-Glutamate (JRHBiosciences), Sodium Pyruvate (Sigma), 2 mercaptoethanol (Sigma) and1000 units/ml recombinant murine GM-CSF (Immunex) as previouslydescribed (26). Day 8 bone marrow-derived DCs were stained withmonoclonal antibodies by conventional methods. Monoclonal antibodyagainst MHC class II, 14-4-4s, was purified from hybridoma supernatant.Dr. William Baldwin, Johns Hopkins University, kindly supplied CTLA4-Igfusion molecule. Antibodies for MHC class I (28-14-8), F4/80 (C1.A3-1),B7.1 (1G10), B7.2 (GL1), FcγRII/III (2.4G2) and Mac-1 (M1/70) werepurchased from PharMingen. For tester cDNA preparation, day 8 cells werepurified by cell sorter using 14-4-4s and CTLA4Ig at the Johns HopkinsUniversity Oncology Center. The purity of MHC class II^(hi) and B7^(hi)population after sorting was 93-98%.

Bone marrow-derived macrophages were cultured with RPMI-1640 mediumsupplemented with 10% FCS, Penicillin/Streptomycin, non-essential aminoacids, sodium pyruvate, L glutamine, 2-mercaptoethanol and 250 units/mlrecombinant murine M-CSF, and they were treated with 500 units/ml γ-IFN(Pharmingen) and 5 μg/ml LPS (Sigma) as previously described (27). Afterstimulation, cell surface expression of MHC class II and B7 wereconfirmed using flow cytometric analysis on day 10 of culture.

Macrophage cell lines WEHI-3, RAW264.7, J774.A.1, PU5-1.8 were kindlyprovided by Dr. Joshua Farber of the MIAID, National Institutes ofHealth. They ware cultured with ATCC recommended medium.

Allogeneic Mixed Lymphocyte Reaction

Day 8 BM-derived DCs characterized as MHC class II^(hi) and B7^(hi) weretested for their ability to stimulate allogeneic T cells in MLC. MLCreactions were performed in 96 well flat bottom microplates by addingincreasing number of BALB/c stimulator cells s to 3×10⁵ allogeneicC57BL/6 lymphocytes. After 3 days of culture, T cell proliferation wasassessed by addition of 1 μCi of [³H]-methyl-thymidine (Amersham) toeach well for the final 18h of culture. Cells were then harvested, andincorporation of radioactivity was determined using a β counter (Packard96).

cDNA Subtractive Hybridization

Total RNA from sorted DCs and activated macrophages was extracted withTRIZOL (Gibco BRL). Messenger RNA was purified by Oligotex mRNApurification hit (Qiagen). We used the PCR based SMART cDNA synthesissystem (Clonetech) to amplify cDNA followed by the PCR based subtractionsystem PCR Select (Clonetech). Subtraction was performed following themanufacturer's protocol. After final subtractive PCR, DNA fragments wereligated into plasmid vectors pCR2.1 (Invitrogen) or pCR Blunt(Invitrogen). After transformation, each clone was grown for plasmid DNAamplification and miniprep DNA and then digested with EcoRI to confirmthe presence of inserts. Plasmid dot blot was then performed to confirmthat the cDNA cloned is dendritic cell specific. Alkaline denaturedminiprep DNAs were spotted on Hybond N+ membrane (Amersham) hybridizedwith SMART cDNA probe-derived from sorted DCs or activated macrophages.These cDNA probes were ³²P labeled using random primer labeling method(Stratagene Prime-It II). Hybridizations and washing were done aspreviously described (28). Membranes were exposed to a film (Amersham)for 1-2 days and developed.

Plasmid Dot Blot Analysis

Alkaline denatured miniprep DHA samples were spotted on Hybond N+membrane (Amersham) and hybridized with SMART® cDNA probe-derived fromsorted DCs or activated macrophages. These cDNA probes were ³²P labeledusing a random primer labeling method (Stratagene Prime-It II).Hybridizations and washing ware done as previously described [cites??].For autoradiography, membranes were exposed to a film (Amersham) for 1-2days and developed.

cDNA Library Construction and Screening—Cloning of B7-DC

Bone marrow-derived DCs were harvested on day 8 without sorting. About20% to 40% of these cells expressed high MHC class II and B7. Total RNAextraction followed by poly A RNA purification was done as describedabove. For oligo dT primed DC library construction, we used Lambda ZAPExpress cDNA synthesis system (Stratagene), the PCR DNA fragment ofB7-DC was probed and used for screening. Membrane transfer,denaturation, renaturation, were performed using Stratagene's protocol.Radiolabeling of probes, hybridization, washing, and autoradiographywere done as described above. Positive clones were isolated and 2ndscreening was performed. After 2nd screening, plasmids were excised byin vivo excision and tested by dot blotting and sequencing. Sequencingwas done by the Core Facility at the Johns Hopkins University School ofMedicine, BLAST program was used to do homology search of the nucleotidesequence against Genbank (NCBI) for similarity to previously reportedgenes. The full length B7-DC cDNA clone was pulled out from the DC cDNAlibrary. 5′ RACE was performed using SMART RACE cDNA amplification kit(Clontech). 5′-RACE products were cloned into pCR2.1 vector andsequenced. Two more full length B7-DC clones were obtained by RT-PCR andtheir sequence were compared to avoid sequence error.

Human B7-DC was cloned as follows: human DCs were obtained from normalperipheral blood mononuclear cells by culture in either GM-CSF+IL-4 orGM-CSF+Flt-3L as described previously (29). RNA was extracted asdescribed above. A BLAST search identified an overlapping EST clone,GenBank accession number AK001879, with homology to mouse B7-DC, 5′ RACEwas performed as described above. We sequenced a 5′-RACE PCR fragmentand designed a primer corresponding to 5′-UTR of human B7DC. Thefollowing primers in the 5′-UTR and 3′-UTR of B7-DC was used to amplifyfull-length human B7-DC:

[SEO ID NO: 6] 5′-GGACCTACTGCATGTTGATTGTTTTG-3′ and [SEQ ID NO: 7]5′-TGCAAACTGAGGCACTGAAAAGTC-3′The full length cDNA sequences of the human and murine B7-DC cDNAs havebeen deposited with EMBL/GenBank/DDBS under accession number AF329193and AF142780.BAC (129SVJ) Library Screening/Genomic Cloning and Mapping

BAC library screening, followed manufacturer's protocol (Genome Systems,Inc.) Primers used:

[SEQ ID NO: 8] 5′-TTGTTGTCTCCTCTGTTCTCCCAAC-3′ and [SEQ ID NO: 9]5′-ACAGTTGCTCCTTGTATCAGGTTC-3′

BAC library screening obtained 3 positive clones. Chromosome locationmapping was done by fluorescence in situ hybridization (Genome SystemsInc.). A total of 80 metaphase cells were analyzed with 79 exhibitingspecific labeling. The human B7-DC mapping was done by using availablebioinformatic tools, NCBI's BLAST program and the International RHMapping Consortium. The hB7-DC sequence was searched in htsg and wasfound to map to two BAC clones RP11-574F11 (AL162253) and Rp11-635N21(AL354744) localizing on chromosome 9.

Virtual Northern Blotting

4-6 weeks old female Balb/c mice were purchased from NCI and used fortissue RNA preparation. RNA extraction and SMART cDNA synthesis fortissues, sorted DCs and activated macrophages were performed asdescribed above. SMART PCR cDNAs were purified by PCR purification kit(Qiagen). 0.5 μg/lane purified DNAs were run on a 1% agarose gel andtransferred on a Nytran nylon membrane (Schleoer and Schuell). To makeradioactive probes, we amplified subtracted library derived plasmid DNAsas templates. We amplified DNA by PCR using primer sets just adjacent tothe cloning site of plasmid DNA and used purified PCR DNA of each of theclones for hybridization probes. The nucleotide sequences of theseprimers are as follows.

[SEQ ID NO: 10] 5′-GTAACGGCCGCCAGTGTGCTG-3′ and [SEQ ID NO: 11]5′-CGCAAGTGTGATGGATATCTGCA-3′Virtual Northern analysis of total RNA of human DCs and control placentawas also performed. The probes used and RNA preparation were describedabove. Radiolabeling of probes, hybridization, washing andautoradiography were done as described above.Hamster Anti-mB7-DC Ab Production

Stable transfectants of B7-DC in DC2.4, RAW246.7 and RENCA cell lineswere used to immunize Armenian Hamsters. The B7-DC was cloned into themodified pCAGGS vector (30). The hamsters were boosted three times withplasmids containing B7-DC (Rockland). The anti-B7-DC antibody used inthis study was from one the sera of one of the three hamsters immunized.

CD28-Ig, CTLA4-Ig and PD-1-Ig Binding Assay

293T cells were transfected with B7.1-pCAGGS, B7-DC-pCAGG, PD-1-pCAGGSor vector alone using Lipofectamine 2000 (Gibco BRL). After 24 hrs,cells were resuspended in FACS buffer (1×HBSS, 2% calf serum, 10 mMHEPES and 0.1% NaN₃) and spun at 1000 rpm for 5 min at 4° C. The bufferwas then decanted, antibody added to the tubes, incubated at 4° C. for20 min, washed 2× with FACS buffer, and repeated this for secondaryantibody. The samples were run on FACScan. B7.1 antibody was used 1:5dilution, 10 μl/sample (Cal-Tag). Recombinant Cd28-Ig, CTLA-4-Ig andPD-1-Ig chimeras were used at 2 μg/ml, 10 μl/sample (R&D System, Inc).Goat F(ab′)₂ anti-human IgG-PE was used at 1:20 dilution (SouthernBiotechnology Associates, Inc.).

B7-DC-Ig Dimer Synthesis

The B7-DC-Ig construct was made by fusing the sequence encoding theN-terminal amino acids of B7-DC without the transmembrane domainin-frame to the sequence encoding the C-terminal amino acids of thehuman IgG₁ Fc in the pIg-Tail Plus vector (R & D systems). COS-7 cellswere transiently transfected with pIg/B7-DC using LipofectAMINE 2000(GIBCO BRL) or GINE JAMMER (Stratagene), The B7-DC-Ig fusion protein waspurified from the serum-free supernatants using the saturated ammoniumsulfate precipitation. SDS-PAGE and silver staining demonstrated apurity >90%.

T Cell Proliferation and Cytokine Assays

For costimulation assays with anti-CD3, 96 well flat bottom plates(Immulon 4 from Dynex) were precoated with anti-CD3 antibodies (2C11,Pharmingen) and B7.1-Ig (R&D System), B7-DC-Ig or Isotype control(Sigma) at 100 ng/ml were diluted in 1×PBS (Gibco) pE 7.4 for two hoursat 37° C. The plates were then washed 3× with 1×PBS and blocked withRPMI1640 medium supplemented with 10% FCS, Penicillin/Streptomycin,non-essential amino acids, sodium pyruvate, L glutamine,2-mercaptoethanol for one half hour before adding T cells. Spleens andlymph nodes were obtained from 6-10 weeks old BALB/c mice. RBCs werelysed using ACK buffer and T cells were purified using dynabeads M-280(Dynex), with the indirect method. The beads were washed 2× with PBS pH7.4+1% FCS before adding the cells and an antibody cocktail composed ofanti-IE^(d) and B220/CD45RO or Cd8α (Pharmingen) was added to the cellsand incubated at 4° C. with bi-directional mixing for 30′. The cellswere isolated by placing the tube in a Dynal MPC for 5′, centrifuged at1500 rpm for 5′ and washed 2× with PBS pH 7.4+1% FCS to remove unboundAbs. The same procedure was repeated with 15′ incubation, and the cellsplated at 2×10⁵ cells/well. After 72 h of incubation, 10 μl of³H-thymidine (1 μCi/well) was added to each well and incubated for 18hrs. Cells were harvested with a Packard Micromate Cell Harvester, andfilters were read on a Packard Matrix 96 direct β counter.

For costimulation assays using the RENCA system to present HA antigen,RENCA cells were cultured with RPMI-1640 medium supplemented with 10%FCS, Penicillin/Streptomycin, non-essential amino acids, sodiumpyruvate. It was induced with IFN-γ (75 U/ml) for 72 hours for MHC classII expression. They were then irradiated for 13,200 Rad. and plated at2×10⁴ cells/well (96 well flat bottom plates). HA110-120 peptide wasthen added at 2.5 ug/well and various concentrations of the Ig-fusionmolecules were added. Transgenic I-E^(d)+HA specific T cells (kind giftof H. von Boehmer, Harvard University) were isolated as described aboveand plated at 4×10⁵ cells/well. After 48 hr of incubation, 10 μl of ³Hthymidine (1 μCi/well) was added to each well and incubated for 18 hrs.Cells were harvested with a Packard Micromate Cell Harvester and filterswere read on a Packard Matrix 96 Direct β counter.

For analysis of cytokine production by ELISA, cultures were set up asdescribed above and supernatants were harvested at the indicated times.IL-2, IL-10 and IPN-γ concentrations were determined using commerciallyavailable ELISA kits (Endogen), and IL-4 and IL-6 (R&D System).

In Vivo Costimulation

Pooled axillary, inguinal, cervical, and mesenteric LNs from the TCRtransgenic mouse line 6.5 that expresses a TCR recognizing an I-E^(d)restricted HA epitope (¹¹⁰SFERFEIFPKE¹²⁰ [SEQ ID NO:12]) on a BI0.D2genetic background were dissociated in RPMI media (GIBCO BRL), passedover 100 μm nylon cell strainer, and washed in sterile Hank's buffer(GIBCO BRL). After FACS® staining to determine the proportion ofclonotypic CD4 cells, a cell preparation containing 2.5×10⁶ clonotypiccells in 0.2 ml sterile Hank's buffer was injected intravenously (i.v.)into the tail vein of recipient BI0.D2 mice. Three days after thisadoptive transfer, the animals were vaccinated via subcutaneous (s.c.)injection into the hind footpads. Each mouse received bilateralinjections of one of three preparations:

-   (A) 10 μg synthetic HA (per footpad) (HA peptide (110-120) combined    in a 1:1 v/v ratio with incomplete Freund's Adjuvant (IFA) (Sigma),-   (B) the HA-IFA mixture with 25 μg of B7-DC-Ig, or-   (C) the HA-IFA mixture with 25 μg of an isotype control antibody. 7    days later, draining LNs nodes were harvested; 1.5×10⁵ LN cells were    incubated in round-bottom 96-well tissue culture plates with the    indicated concentration of synthetic HA peptide. Proliferation    assays were performed by pulsing 48 h cultures with 1 μCi    [³H]thymidine and incubating an additional 12 h before harvest and    determination of the amount of incorporated radioactivity.

EXAMPLE II Identification and Characterization of B7-DC

B7-DC was isolated from a subtracted library between DCs and activatedmacrophages. The two populations used for cDNA subtraction were bonemarrow-derived GM-CSF cultured DCs as the “tester” population andγ-interferon+LPS activated adherent bone marrow-derived M-CSFmacrophages as the “driver” population. Day 8 MHC class II^(hi)B7^(hi)“mature” DCs were sorted to >93% purity as the source of tester cDNA.DCs were characterized by flow cytometry as having roughly 50 foldhigher MHC class II levels than macrophages. Both populations expressedB7.1 and B7.2 although B7.2 levels were significantly higher in the DCs.F4/80 and CD16 were expressed at higher levels on the macrophagepopulation. Functional comparison of the two populations demonstratedthat the DC population was roughly 100 fold more potent than themacrophage population in stimulating an allogeneic mixed lymphocytereaction.

After RNA extraction from both populations, we used a PCR based cDNAsynthesis system followed by the PCR based subtraction procedure, PCRSelect. One of the differentially expressed clones encoded a novelimmunoglobulin supergene family member, which we name B7-DC. The murineB7-DC cDNA is ˜1.7 kb in length encoding a 247 amino acid (aa) precursorprotein with a 23 aa N-terminal signal peptide and a predicted molecularweight of ˜25 kd (Table 1). The putative leader sequence andtransmembrane domain were identified using the SOSUI program (31). Twocharged aa are found within the 23 aa transmembrane domain of mB7-DC,suggesting a possible binding partner. At the aa level, murine B7-DC is70% identical to the human B7-DC indicating that they are orthologues(See Tables below)

TABLE 1 Amino acid sequence comparison of murine B7-DC and human B7-DC.    Puatative leader sequence mB7-DCMLLLLPILNLSLQLHPVAALFTVTAPKEVYTVDvGSSVSLECDFDRRECTELEGIRASLQ hB7-DCMIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQ*::** :*.*.**** :*******.***:* :: **.*:***:**  . .:* .* **** mB7-DCKVENDTSLQSERATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVICGAAWDYKYLTVKVK hB7-DCKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK******* : *************** ****.*****.***:*::* *.********:*** mB7-DCASYMRIDTRILEVPGTGEVQLTCQARGYPLAEVSWQNVSVPANTSHIRTPEGLYQVTSVL hB7-DCASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVL*** :*:*:**:** *.**:***** ********* ********** *************                                            Putative TM domain mB7-DCRLKPQPSRNFSCMFWNAHMKELTSAIIDPLSRMEPKVPRTWPLHVFIPACTIALIFLAIV hB7-DCRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPSCIIAFIFIATV**** *.*****:***:*::*** * **  *:***:.  ** **:***:* **:**:* * mB7-DCIIQRKRI-------------------------- hB7-DCIALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI *  **:: The mB7-DC putative leader andtransmembrane domain are overlined. The alignment was done usingClustalw-Gonnet Pam250 matrix. The [*] indicates identical amino acidsanD [:] shows conservative substitutions. Cysteine residues that may beimportant in the formation of disulfied bonds inside the immunoglobutinV or C domains are italicized.

TABLE 2  Amino acid sequence comparison of mB7-DC and mB7-H1 mB7-DCMLLLLPILNLSLQLHPVAALFTVTAPKEVYTVDVGSSVSLECDFDRRECTELEGIRASLQ mB7-H1-MRIFAGIIFTACCH-LLRAFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWE : ::. : ::   * :   **:****::*.*: **.*::** *  ..  :* .: .  : mB7-DCK----------VENDTSLQSE----RATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVIC mB7-H1KEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISY*           *:* . * .    **:* ::**  *:* ::*..*:::*:* * *:: mB7-DCGAAWDYKYLTVKVKASYMRIDTRILEVPGTGEVQLTCQARGYPLAEVSWQN-----VSVP mB7-H1GGA-DYKRITLKVNAPYRKINQRISVdPATSEHELICQAEGYPEAEVIWTNSDHQPVSGK*.* *** :*:**:*.* :*: **   *.*.* :* ***.*** *** * *     ** mB7-DCANTSHIRTPEGLYQVTSVLRLKPQPSRNFSCMFWNAH--MKELTSAIIDPLSRMEPKVPR mB7-H1RSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNR ..:  **   * :*** **::. ..  * * **.::   :. :. **  *.  .*   * mB7-DCT-WPLHVFIPACTIALIFLAIVIIQRKRI------------------------ mB7-H1THWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET* * *   *    *.:  : :.: :: *:The hB7-DC differs slightly from the murine B7-DC in that it has alonger cytoplasmic tail.,

TABLE 3 Amino acid sequence comparison of B7-DC to the B7 familymembers. Reference B7 Protein Compared to: % identity¹ % similarity²mB7-DC hB7-DC 70 80 hBT3.1 25 41 mB7-DC mB7-H1 34 48 mBT⁴ 30 45 mB7.1 — —³ mB7.2 — — mB7RP-1/B7h — — MBT mB7.1 24 48 mB7.2 24 40 MB7.1 mB7.2 2745 MB7-H1⁵ mB7.1 23 40 mB7.2 25 49 mB7RP-1/B7h 24 41 mBT 24 45Comparison were done using NCBI blast2 search (matrix BLOSUM62).¹Identical amino acids at corresponding positions ²Similar amino acidsat corresponding position - grouped as follows: (A, G); (S, T); (E, D);(R, K, H); (Q, N); (V, I, L, M); (Y, F); (W); (C); (P) ³no significantsimilarity was found using matrix BLOSUM62 ⁴BT = butyrophilin ⁵= PDL-1

Through a homology search, it was found that B7-DC has significanthomology to B7-H1 (34% identity, 48% similarity) (Table 2), to a lesserextent butyrophilin (30% identity, 45% similarity), and <20% identity toB7.1 and B7.2 (Table3). Phylogenetic studies indicate that butyrophilinis likely related to the B7 family through exon shuffling (32, 33). Theyeach possess the canonical IgV-IgC structure and a transmembrane domain.In contrast to the other B7 family members, murine B7DC has an extremelyshort cytoplasmic tail (4 aa).

To determine the genomic structure of mB7-DC, the present inventorsisolated a genomic clone by screening a pooled Bacterial ArtificialChromosome (BAC) library using probes from the 5′ and 3′ UTRs.Chromosome location mapping was performed using the BAC clones.Chromosome localization of B7-DC was done using florescent in situhybridization (FISH). Measurements of 10 specifically labeledchromosomes 19 demonstrated that mB7-DC is located at a position whichis 47% of the distance from the heterochromtic-euchromatic boundary tothe telomere of chromosome 19, an area that corresponds to the interfacebetween bands 19C2 and 19C3. Specific hybridization signals weredetected by incubating the hybridized slides in fluoresceinatedantidigoxigenin antibodies followed by counter staining with DAPI. Thislocus corresponds to a region of human chromosome 9, where hB7-H1 hasbeen mapped.

hB7-DC was found to be located on two chromosome 9 BAC clones. Inaddition, both hB7-DC and hB7-H1 were found to be located on a singlechromosome 9 BAC clone with an insertion size of approximately 164 kb(FIG. 1). The genomic proximity of B7-DC and B7-H1 is reminiscent of theB7.1/B7.2 pair, which map to within one megabase of each other.

EXAMPLE III B7-DC is Selectively Expressed in Dendritic Cells

In order to determine the expression pattern of B7-DC, virtual Northernanalysis was performed using RNA extracted from multiple tissues,macrophage cell lines, macrophage cultures and dendritic cells derivedfrom both bone marrow and spleen. While strong hybridization wasdetected using a B7-DC probe in immature (day 4,6) and mature (day 8 andsorted MHC II^(hi)B7^(hi)) bone marrow derived DC and splenic DC, nosignal was detected in any of 4 macrophage cell lines, activated BMmacrophages or peritoneal macrophages (FIG. 2). Strong expression ofhB7-DC was detected in human DCs grown from peripheral blood mononuclearcells with GM-CSF plus either IL-4 or Flt-3L (FIG. 3). In order toverify cell surface expression of B7-DC protein, anti-mB7-DC antibodieswere used to stain DCs. Staining, blockable with soluble B7-DC-Ig, wasobserved on DC (FIG. 4).

B7-DC does not Bind to CD28 or CTLA-4 but does Bind to PD-1

Although B7-DC has structural and sequence homology to the B7 family, itdoes not contain the putative CD28/CTLA-4 binding sequence, SQDXXXELY[SEQ ID NO:13] or XXXYXKRT [SEQ ID NO:14] (34) (where X=any amino acid).To directly assess binding, the ability of dimeric CD28-Ig and CTLA-4-Igto stain 293T cells transfected with either B7-DC or B7.1 wasinvestigated. Whereas strong binding was observed with B7.1transfectants, there was no binding to B7-DC transfectants (FIG. 5).Based on homology and genomic proximity between B7-DC and B7-H1/PD-L1,experiments were conducted to test PD-1 as a candidate binding partnerfor B7-DC. Ideed, PD-1001G bbound to B7-DC transfectants but not to B7.1transfectants. The binding of BPD-1-Ig too B7-DC transfectants was lowerthan thg bidig of CTL-4-It and CB28-Ig to B7.1 transfectants, althoughit was specific. Further confirmation of the bnding of PD-1 to B7-Dc wasotained from positive stainig of stablee B7-DC-GFP transfectants withPD-1-Ig. It was concluded that, as with B7-H1 and B7h/B7RP-1, B7-DC doesnot use CD28 or CTLA-4 as receptors. Rather, PD-1 appears to be areceptor for B7-DC.

EXAMPLE IV B7-DC Functions as a Costimulatory Molecule for T Cells

A soluble B7-DC-Ig fusion protein which could be added to T cellstimulation assays was produced for use in testing whether B7-DCpossessed costimulatory activity. The proliferative response of T cellswas measured to stimulation by increasing amounts of immobilizedanti-CD3 in the presence of either B7-DC-Ig, B7.1-Ig or an isotypecontrol. FIG. 6 (left) shows that, in the presence of suboptimalanti-CD3 concentration, B7-DC costimulated a greater T cellproliferative response than did B7.1. Furthermore, B7-DC costimulatedproliferative responses were higher in CD4 than in CD8 cells (FIG. 6,right). B7-DC failed to stimulate T cells in the absence of aTCR-focused stimulus, indicating that B7-DC was providing a truecostimulatory signal.

B7-DC also costimulated a proliferative responses when “signal 1” wasprovided by an MHC-peptide complex. RENCA cells (which express noendogenous B7.1, B7.2 or B7-DC by RT-PCR analysis) were treated withγ-IFN to induce MHC class II expression. These cells were loaded withthe I-E^(d) restricted HA 110-120 peptide (FERFEIFPKE)(35) [SEQ IDNO:15]. Purified splenic T cells from an I-E^(d)+HA 110-120 specific TCRtransgenic mouse line were added and the proliferative response wasmeasured in the presence of either B7-DC-Ig, B7.1-Ig or an isotypecontrol. FIG. 7 shows that B7-DC possessed greater costimulatoryactivity than did B7.1.

Patterns of Lymphokine Production Costimulation by B7-DC

The best characterized T cell responses to costimulation by the B7family molecules is lymphokine production. These lymphokines areimportant mediators of T cell effects. Studies were done to analyzeproduction of a number of different lymphokines by T cells that had beenstimulated with anti-CD3 or HA antigen (FIG. 8) costimulated with eitherB7-DC-Ig, B7.1-Ig or an isotype control. Patterns of lymphokinecostimulation were fairly consistent whether anti-CD3 or an MHC-peptidecomplex was utilized as “signal 1”. Significantly, B7-DC costimulatedgreater levels of γ-IFN than did B7.1. B7-DC also costimulatedsignificant amounts of IL-6 production whereas B7.1 T costimulatedvirtually none. While both molecules costimulated IL-2 production, B7-DCdid so more efficiently than did B7-DC. Thus, the patterns ofcostimulation by B7-DC and B7.1 are distinct, with B7-DC being moreefficient in costimulating important proinflammatory lymphokines.

EXAMPLE V B7-DC Enhances In Vivo Immune Responses

In order to determine whether B7-DC possesses in vivo biologic activity,the inventors asked whether B7-DC-Ig enhanced immune responses topeptide vaccines. B7-DC-Ig or an isotype control antibody was added tothe immunogenic cocktail of HA 110-120 peptide and IFA. To permitenumeration of HA-specific CD4 T cells in vivo, 2.5×10⁶ anti-HA 6.5 Tcells were transferred into the mice 3 days before immunization. Sevendays after immunization, draining LN cells were harvested and the cellsstimulated in vitro for 2 days with varying amounts of HA110-120peptide. FIG. 9 shows that addition of B7-DC-Ig indeed dramaticallyenhanced the proliferative response to HA. The total number ofHA-specific T cells in draining LN was increased by roughly 2 fold ingroups receiving B7-DC-Ig relative to isotype antibody controls. It wastherefore concluded that B7-DC had the ability to enhanceantigen-specific responses even on a per cell basis.

EXAMPLE VI Discussion and Conclusions

The present inventors have discovered and characterized a new B7 familymember with expression highly restricted to DCs and having uniquecostimulatory properties for T cells. The human orthologue of B7-DC isalso expressed in DCs.

This restricted expression pattern contrasts with the previouslydescribed B7 family members, suggesting that B7-DC participates indifferent immune responses than the known B7.1/2 pathways. While a weakB7-DC signal was detected by RT-PCR in activated macrophages,preliminary realtime RT-PCR analysis indicated that B7-DC mRNAexpression in DCs was >15-fold higher than in activated macrophages.Antibody staining likewise detected very low levels of B7-DC on thesurface of activated macrophages. It is unclear whether this issufficient for significant T cell activation.

The unusual pattern of lymphokine production that B7-DC costimulatesimplies a unique biologic role compared to other B7 family members. Thetraditional classification of cytokines is as follows: Th1cytokinesinclude IL-2, γ-IFN and lymphotoxin; Th2 cytokines include IL-4, IL-5,IL-6 and IL-13 (36), B7-DC does not induce either a classic Th1 or Th2lymphokine profile. B7-DC induces very little IL-4 and no IL-10.However, IL-6 is considered a Th2 cytokine. The lower IL-2 and higherγ-IFN costimulated by B7-DC relative to B7.1 does not conform to aclassic Th1 pattern. Nonetheless, the high γ-IFN production suggeststhat B7-DC evokes important T cell effector function.

B7-DC is noteworthy in its ability to costimulate IL-6. The robustproliferative response of T cells induced by B7-DC is explained in partby its strong costimulation of IL-6 production, which is not observedwith B7.1. IL-6 is a potent amplifier of T cell proliferation (inconjunction with other proliferative stimuli) (37,38). IL-6 is amultifunctional cytokine that regulates not only T cell function butalso proinflammatory responses, monocyte differentiation, B celldifferentiation, thrombopoiesis, bone resorption, and the growth ofcertain hematopoietic tumors (39, 40). IL-6 can function in concert withsoluble IL6 receptors (sIL-6 R) in the induction of chemokines andleukocyte recruitment (41). It can mediate potent antiapoptotic effectsvia Stat-3 activation. IL-6 dependent Stat-3 activation in T cells hasbeen reported to be an important pathway for the survival of activated Tcells (42, 43) although, other reports suggest that Stat-3 exerts itseffect on resting T cells.

While B7-DC fails to bind CD28 or CTLA-4, it does bind PD-1, a receptorfor B7-H1/PD-L1 (22, 47, 48). It has not yet been determined whether itbinds ICOS, a receptor for B7 h/B7RP-1 (23-25, 44-46). The markedhomology between B7-DC and B7-H1/PDL-1 (greater than that between B7.1and B7.2), the close physical linkage of hB7-H1/PD-L1 and hB7-DC andtheir binding to a common receptor, suggests that they are related by arelatively recent gene duplication event. This is analogous to therelationship between B7.1 and B7.2, which both map to within onemegabase on mouse chromosome 16 and on human chromosome 3 (49).

It will be important to discern the relative biologic roles of B7-DC vsB7-H1/PD-L1 as mediated by PD-1 and other putative receptor(s). PD-1 isexpressed subsequent to, and appears to inhibit, T cells activation.PD-1 induces apoptosis under conditions of T cell stimulation with highconcentrations of anti-CD3. PD-1 knockout mice develop an autoimmunesyndrome (22) characterized by clinical manifestations of hypertropiccardiomyopathy. In contrast, Dong et al (21) reported that B7-H1/PD-L1co-stimulated T cell proliferation and cytokine release at lowerconcentrations of anti-CD3. By analogy to the relationship of CD28/CTLA-4, PD-L1 may be a counterreceptor for an as yet unidentifiedactivating receptor. Despite sharing the property of binding to PD-1,B7-DC and B7-H1 are distinct in their lymphokine costimulation patterns;B7-H1 costimulated T cell IL-10 production whereas B7-DC does not. Thedistinct cellular expression patterns and costimulatory functions ofB7-DC suggest a unique role in immune function.

The references cited above and below are all incorporated by referenceherein, whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

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in addition to documents cited fully in the text, certain documents arecited by number only (parenthetical); the latter are listed below.

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What is claimed is:
 1. A method of treating a human subject havingcancer comprising administering to the subject: a) a tumor-reducingregimen of chemotherapy, irradiation or surgical resection; and b) apharmaceutical composition comprising a fusion protein comprising afirst fusion partner consisting of amino acids 20-221 of SEQ ID NO:2 anda second fusion partner consisting of the hinge, CH2 and CH3 regions ofa human immunoglobulin C 1 chain.
 2. The method of claim 1 wherein thefusion protein comprises a linker between the first and second fusionpartners.
 3. The method of claim 1 wherein the first fusion partner isdirectly linked to the second fusion partner.
 4. The method of claim 1 ,wherein the fusion protein is administered as a dimer formed by covalentbonding of Cys residues in at least one of said CH2 and CH3.
 5. Themethod of claim 1 wherein the cancer is a melanoma.
 6. The method ofclaim 1 wherein the cancer is a carcinoma.
 7. The method of claim 6wherein the carcinoma is hepatocellular carcinoma.
 8. The method ofclaim 6 wherein the carcinoma is cervical cancer.
 9. The method of claim8 wherein the cervical cancer is caused by human papillomavirus (HPV).10. The method of claim 6 wherein the subject has a chronic human viralinfection.
 11. The method of claim 10 wherein the chronic human viralinfection is due to a virus selected from the group consisting ofhepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiencyvirus (HIV), Epstein Barr virus (EBV) and herpes simplex virus (HSV).12. The method of claim 6 wherein the subject has a non-viral infectiousdisease.
 13. The method of claim 2 wherein the pharmaceuticalcomposition is administered to the human in an effective amount toinduce, enhance, increase, or potentiate an immune response against thecancer.
 14. The method of claim 2 further comprising administering tothe human a general immunostimulatory agent or adjuvant.
 15. The methodof claim 14 wherein the general immunostimulatory agent or adjuvant ispart of the pharmaceutical composition.
 16. A method of treating a humansubject having cancer comprising administering to the subject apharmaceutical composition comprising a fusion protein comprising afirst fusion partner consisting of amino acids 20-221 of SEQ ID NO:2 anda second fusion partner consisting of the hinge, CH2 and CH3 regions ofa human immunoglobulin C 1 chain in combination with a second treatmentselected from the group consisting of chemotherapy, irradiation, andsurgical resection.
 17. The method of claim 16 wherein thepharmaceutical composition is administered to the subject aftertreatment of the subject with the chemotherapy, irradiation, or surgicalresection.
 18. The method of claim 16 wherein the pharmaceuticalcomposition is administered to the subject before treatment of thesubject with the chemotherapy, irradiation, or surgical resection. 19.The method of claim 16 wherein the pharmaceutical composition isadministered to the subject simultaneously with the chemotherapy,irradiation, or surgical resection.
 20. A method of increasing an immuneresponse in a human subject having cancer comprising administering tothe subject a pharmaceutical composition comprising a fusion proteincomprising a first fusion partner consisting of amino acids 20-21 of SEQID NO:2 and a second fusion partner consisting of the hinge, CH2 eandCH3 regions of a human immunoglobulin C 1 chain in combination with asecond treatment selected from the group consisting of a tumor-reducingregimen of chemotherapy, irradiation, and surgical resection.
 21. Themethod of claim 20 wherein the immune response is against the cancer.22. The method of claim 21 wherein the cancer is a melanoma or acarcinoma.